MENU
The Electronic Scholarly Publishing Project: Providing world-wide, free access to classic scientific papers and other scholarly materials, since 1993.
More About: ESP | OUR CONTENT | THIS WEBSITE | WHAT'S NEW | WHAT'S HOT
ESP: PubMed Auto Bibliography 10 Feb 2025 at 01:44 Created:
CRISPR-Cas
Clustered regularly interspaced short palindromic repeats (CRISPR, pronounced crisper) are segments of prokaryotic DNA containing short repetitions of base sequences. Each repetition is followed by short segments of "spacer DNA" from previous exposures to foreign DNA (e.g a virus or plasmid). The CRISPR/Cas system is a prokaryotic immune system that confers resistance to foreign genetic elements such as those present within plasmids and phages, and provides a form of acquired immunity. CRISPR associated proteins (Cas) use the CRISPR spacers to recognize and cut these exogenous genetic elements in a manner analogous to RNA interference in eukaryotic organisms. CRISPRs are found in approximately 40% of sequenced bacterial genomes and 90% of sequenced archaea. By delivering the Cas9 nuclease complexed with a synthetic guide RNA (gRNA) into a cell, the cell's genome can be cut at a desired location, allowing existing genes to be removed and/or new ones added. The Cas9-gRNA complex corresponds with the CAS III crRNA complex in the above diagram. CRISPR/Cas genome editing techniques have many potential applications, including altering the germline of humans, animals, and food crops. The use of CRISPR Cas9-gRNA complex for genome editing was the AAAS's choice for breakthrough of the year in 2015.
Created with PubMed® Query: ( "CRISPR.CAS" OR "crispr/cas" ) NOT pmcbook NOT ispreviousversion
Citations The Papers (from PubMed®)
RevDate: 2025-02-07
CmpDate: 2025-02-08
Challenges in developing a split drive targeting dsx for the genetic control of the invasive malaria vector Anopheles stephensi.
Parasites & vectors, 18(1):46.
BACKGROUND: Anopheles stephensi is a competent malaria vector mainly present in southern Asia and the Arabian Peninsula. Since 2012, it has invaded several countries of eastern Africa, creating an emerging risk of urban transmission. Urgent efforts are required to develop novel and more efficient strategies for targeted vector control. CRISPR/Cas9-based homing gene drives have been proposed as attractive alternative strategies. Gene drives have the potential to spread a desired trait through a population at higher rates than via normal Mendelian inheritance, even in the presence of a fitness cost. Several target genes have been suggested and tested in different mosquito vector species such as Anopheles gambiae and Aedes aegypti. Several promising suppression drives have been developed in An. gambiae that target the sex determination gene doublesex (dsx).
METHODS: In this study, a geographically confineable gene drive system targeting dsx was developed (dsx[gRNA]). Here, a transgenic line which expresses Cas9 under the control of the endogenous zpg promoter was generated. Separately a transgenic line which expresses a gRNA targeting the female specific exon of dsx was inserted into that same target site. The reproductive fitness of males and females heterozygous and homozygous for this element was determined. A series of experimental crosses was performed to combine the two elements and assess the homing rate of the dsx element in a split drive system.
RESULTS: The drive was able to home in a super-Mendelian rate comparable to those obtained by an autonomous drive in this species. Although inheritance rates as high as 99.8% were observed, potentially providing very potent gene drive, dominant effects on male and female fertility were observed, which would be sufficient to hinder spread of such a drive. Molecular analysis indicated that the gRNA expressing insertion disrupted normal splicing of dsx.
CONCLUSIONS: These results should be considered when proposing the viability of dsx as a target gene for a population suppression gene drives in Anopheles stephensi. Although high homing rates were observed, the fitness defects found in both males and females carrying the transgene would likely prohibit this drive from functioning in the field.
Additional Links: PMID-39920845
PubMed:
Citation:
show bibtex listing
hide bibtex listing
@article {pmid39920845,
year = {2025},
author = {Larrosa-Godall, M and Ang, JXD and Leftwich, PT and Gonzalez, E and Shackleford, L and Nevard, K and Noad, R and Anderson, MAE and Alphey, L},
title = {Challenges in developing a split drive targeting dsx for the genetic control of the invasive malaria vector Anopheles stephensi.},
journal = {Parasites & vectors},
volume = {18},
number = {1},
pages = {46},
pmid = {39920845},
issn = {1756-3305},
support = {INV-008549/GATES/Bill & Melinda Gates Foundation/United States ; BBS/E/I/00007033, BBS/E/I/00007038, and BBS/E/I/00007039/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; },
mesh = {Animals ; *Anopheles/genetics/physiology ; *Mosquito Vectors/genetics ; Female ; Male ; *Gene Drive Technology/methods ; *Malaria/transmission/prevention & control ; *Mosquito Control/methods ; *Animals, Genetically Modified/genetics ; *CRISPR-Cas Systems ; Sex Determination Processes/genetics ; },
abstract = {BACKGROUND: Anopheles stephensi is a competent malaria vector mainly present in southern Asia and the Arabian Peninsula. Since 2012, it has invaded several countries of eastern Africa, creating an emerging risk of urban transmission. Urgent efforts are required to develop novel and more efficient strategies for targeted vector control. CRISPR/Cas9-based homing gene drives have been proposed as attractive alternative strategies. Gene drives have the potential to spread a desired trait through a population at higher rates than via normal Mendelian inheritance, even in the presence of a fitness cost. Several target genes have been suggested and tested in different mosquito vector species such as Anopheles gambiae and Aedes aegypti. Several promising suppression drives have been developed in An. gambiae that target the sex determination gene doublesex (dsx).
METHODS: In this study, a geographically confineable gene drive system targeting dsx was developed (dsx[gRNA]). Here, a transgenic line which expresses Cas9 under the control of the endogenous zpg promoter was generated. Separately a transgenic line which expresses a gRNA targeting the female specific exon of dsx was inserted into that same target site. The reproductive fitness of males and females heterozygous and homozygous for this element was determined. A series of experimental crosses was performed to combine the two elements and assess the homing rate of the dsx element in a split drive system.
RESULTS: The drive was able to home in a super-Mendelian rate comparable to those obtained by an autonomous drive in this species. Although inheritance rates as high as 99.8% were observed, potentially providing very potent gene drive, dominant effects on male and female fertility were observed, which would be sufficient to hinder spread of such a drive. Molecular analysis indicated that the gRNA expressing insertion disrupted normal splicing of dsx.
CONCLUSIONS: These results should be considered when proposing the viability of dsx as a target gene for a population suppression gene drives in Anopheles stephensi. Although high homing rates were observed, the fitness defects found in both males and females carrying the transgene would likely prohibit this drive from functioning in the field.},
}
MeSH Terms:
show MeSH Terms
hide MeSH Terms
Animals
*Anopheles/genetics/physiology
*Mosquito Vectors/genetics
Female
Male
*Gene Drive Technology/methods
*Malaria/transmission/prevention & control
*Mosquito Control/methods
*Animals, Genetically Modified/genetics
*CRISPR-Cas Systems
Sex Determination Processes/genetics
RevDate: 2025-02-09
CmpDate: 2025-02-09
CRISPR/Cas9-mediated knockout of Tektin 4-like gene (TEKT4L) causes male sterility of Cydia pomonella.
Insect biochemistry and molecular biology, 177:104257.
The sterile insect technique (SIT) is a well-established and environmentally benign method for population control. Identifying genes that regulate insect fertility while preserving growth and development is crucial for implementing a novel SIT-based pest management approach utilizing CRISPR/Cas9 to target these genes for genetic manipulation. Tektin (TEKT), an essential alpha-helical protein pivotal in sperm formation due to its role in cilia and flagella assembly, has garnered attention. In this study, we identified 7 TEKT genes in the testis of Cydia pomonella, a globally invasive fruit pest. Notably, Tektin4-like (TEKT4L) displayed the highest expression level in male adult especially the testes, suggesting its significance in reproductive processes. By utilizing CRISPR/Cas9 technology to knockout TEKT4L, male sterility was induced, showcasing dominant inherited. When wild-type (WT) females mated with TEKT4L[-/-] males, eggs laying proceeded normally, but the hatching rate was dramatically reduced, with only 15.49% progressing to the eyespot stage and 68.86% failing to develop normally. The reproductive fitness of TEKT4L[-/-] males was robust enough to facilitate the transmission of genetic modifications efficiently within the C.pomonella population, yielding a small number of viable offspring. Subsequent cage trials demonstrated the effectiveness of this population in suppressing laboratory populations of C.pomonella, achieving notable results with a relatively low release ratio (TEKT4L[-/-]♂: WT♂: WT♀ = 5:1:5). Consequently, the targeted disruption of the TEKT4L gene holds promise as a fundamental element in a novel pest control strategy against C. pomonella.
Additional Links: PMID-39756499
Publisher:
PubMed:
Citation:
show bibtex listing
hide bibtex listing
@article {pmid39756499,
year = {2025},
author = {Wei, Z and Wang, C and Zhang, X and Lv, Y and Li, Y and Gao, P and Yang, X},
title = {CRISPR/Cas9-mediated knockout of Tektin 4-like gene (TEKT4L) causes male sterility of Cydia pomonella.},
journal = {Insect biochemistry and molecular biology},
volume = {177},
number = {},
pages = {104257},
doi = {10.1016/j.ibmb.2025.104257},
pmid = {39756499},
issn = {1879-0240},
mesh = {Animals ; Male ; *CRISPR-Cas Systems ; *Infertility, Male/genetics ; *Moths/genetics/growth & development ; *Insect Proteins/genetics/metabolism ; Gene Knockout Techniques ; Female ; Testis/metabolism ; Microtubule Proteins ; },
abstract = {The sterile insect technique (SIT) is a well-established and environmentally benign method for population control. Identifying genes that regulate insect fertility while preserving growth and development is crucial for implementing a novel SIT-based pest management approach utilizing CRISPR/Cas9 to target these genes for genetic manipulation. Tektin (TEKT), an essential alpha-helical protein pivotal in sperm formation due to its role in cilia and flagella assembly, has garnered attention. In this study, we identified 7 TEKT genes in the testis of Cydia pomonella, a globally invasive fruit pest. Notably, Tektin4-like (TEKT4L) displayed the highest expression level in male adult especially the testes, suggesting its significance in reproductive processes. By utilizing CRISPR/Cas9 technology to knockout TEKT4L, male sterility was induced, showcasing dominant inherited. When wild-type (WT) females mated with TEKT4L[-/-] males, eggs laying proceeded normally, but the hatching rate was dramatically reduced, with only 15.49% progressing to the eyespot stage and 68.86% failing to develop normally. The reproductive fitness of TEKT4L[-/-] males was robust enough to facilitate the transmission of genetic modifications efficiently within the C.pomonella population, yielding a small number of viable offspring. Subsequent cage trials demonstrated the effectiveness of this population in suppressing laboratory populations of C.pomonella, achieving notable results with a relatively low release ratio (TEKT4L[-/-]♂: WT♂: WT♀ = 5:1:5). Consequently, the targeted disruption of the TEKT4L gene holds promise as a fundamental element in a novel pest control strategy against C. pomonella.},
}
MeSH Terms:
show MeSH Terms
hide MeSH Terms
Animals
Male
*CRISPR-Cas Systems
*Infertility, Male/genetics
*Moths/genetics/growth & development
*Insect Proteins/genetics/metabolism
Gene Knockout Techniques
Female
Testis/metabolism
Microtubule Proteins
RevDate: 2025-02-09
CmpDate: 2025-02-09
β2-tubulin and its promoter in the brown planthopper: A versatile tool for genetic control strategies.
Insect biochemistry and molecular biology, 177:104244.
At present, the application of CRISPR/Cas9 technology for genetic manipulation in insects is predominantly concentrated on Diptera model species, including Drosophila and mosquitoes. In contrast, non-model insects such as the brown planthoppers (BPH, Nilaparvata lugens), a major insect pest of rice, have received less attention in genetic manipulation due to insufficient tools. Here, the analysis of spatiotemporal expression patterns revealed that β2-tubulin in BPH (NlB2t) was predominantly concentrated in male adults and male testis, exhibiting high expression levels. Knockdown of NlB2t expression by using RNAi resulted in the obstruction of male testis development. Mating between the RNAi-treated males and wild-type females led to a notable reduction in the number of eggs laid and the hatching rate of those eggs by 58.2% and 50.6%, respectively. The longevity of RNAi males significantly increased, and females that had previously mated with RNAi males exhibited a diminished inclination for re-mating with wild-type males. The dual-luciferase reporter assay demonstrated robust promoter activity in the upstream 943 bp of NlB2t, capable of driving Cas9 protein expression in vivo and effectively inducing target gene knockout. These findings elucidated that NlB2t may be a key gene in BPH male testis development and reproduction, as a promising target for sterilization. Its upstream promoter serves as a germline promoter, significantly facilitating the development of genetic control tools based on CRISPR/Cas9 technology in BPH.
Additional Links: PMID-39674516
Publisher:
PubMed:
Citation:
show bibtex listing
hide bibtex listing
@article {pmid39674516,
year = {2025},
author = {Chen, JX and Zhang, CC and Sun, JW and Zhang, YB and Luo, MS and Zhang, WQ},
title = {β2-tubulin and its promoter in the brown planthopper: A versatile tool for genetic control strategies.},
journal = {Insect biochemistry and molecular biology},
volume = {177},
number = {},
pages = {104244},
doi = {10.1016/j.ibmb.2024.104244},
pmid = {39674516},
issn = {1879-0240},
mesh = {Animals ; *Hemiptera/genetics/metabolism/growth & development ; Male ; Female ; *Promoter Regions, Genetic ; *Tubulin/genetics/metabolism ; Insect Proteins/genetics/metabolism ; Testis/metabolism ; CRISPR-Cas Systems ; RNA Interference ; },
abstract = {At present, the application of CRISPR/Cas9 technology for genetic manipulation in insects is predominantly concentrated on Diptera model species, including Drosophila and mosquitoes. In contrast, non-model insects such as the brown planthoppers (BPH, Nilaparvata lugens), a major insect pest of rice, have received less attention in genetic manipulation due to insufficient tools. Here, the analysis of spatiotemporal expression patterns revealed that β2-tubulin in BPH (NlB2t) was predominantly concentrated in male adults and male testis, exhibiting high expression levels. Knockdown of NlB2t expression by using RNAi resulted in the obstruction of male testis development. Mating between the RNAi-treated males and wild-type females led to a notable reduction in the number of eggs laid and the hatching rate of those eggs by 58.2% and 50.6%, respectively. The longevity of RNAi males significantly increased, and females that had previously mated with RNAi males exhibited a diminished inclination for re-mating with wild-type males. The dual-luciferase reporter assay demonstrated robust promoter activity in the upstream 943 bp of NlB2t, capable of driving Cas9 protein expression in vivo and effectively inducing target gene knockout. These findings elucidated that NlB2t may be a key gene in BPH male testis development and reproduction, as a promising target for sterilization. Its upstream promoter serves as a germline promoter, significantly facilitating the development of genetic control tools based on CRISPR/Cas9 technology in BPH.},
}
MeSH Terms:
show MeSH Terms
hide MeSH Terms
Animals
*Hemiptera/genetics/metabolism/growth & development
Male
Female
*Promoter Regions, Genetic
*Tubulin/genetics/metabolism
Insect Proteins/genetics/metabolism
Testis/metabolism
CRISPR-Cas Systems
RNA Interference
RevDate: 2025-02-09
CmpDate: 2025-02-09
SYNCAS based CRISPR-Cas9 gene editing in predatory mites, whiteflies and stinkbugs.
Insect biochemistry and molecular biology, 177:104232.
Despite the establishment of CRISPR-Cas9 gene editing protocols in a wide range of organisms, genetic engineering is still challenging for many organisms due to constraints including lethality of embryo injection, difficulties in egg/embryo collection or viviparous lifestyles. Recently, an efficient CRISPR-Cas9 method, termed SYNCAS, was developed to genetically modify spider mites and thrips species. The method is based on maternal injection of formulated CRISPR-Cas9 using saponin and BAPC. Here, we investigate whether the method can be used to perform gene editing in other arthropods such as the beneficial predatory mites Amblyseius swirskii and Phytoseiulus persimilis, and the pests Bemisia tabaci and Nezara viridula. For the predatory mites, Antp and SLC25A38 were used as target genes, while the ortholog of the Drosophila melanogaster ABCG transporter white was targeted in B. tabaci and N. viridula. All species were successfully edited with the highest efficiencies (up to 39%) being obtained for B. tabaci. For A. swirskii and P. persimilis no clear phenotypes could be observed, even though SLC25A38 was successfully knocked-out. The lack of a color phenotype in SLC25A38 mutants was confirmed in the spider mite Tetranychus urticae. Disruption of the target gene Antp is likely lethal in predatory mites, as no true null mutants could be recovered. For B. tabaci, KO of white resulted in orange eyes which diverges from the phenotype seen in white mutants of D. melanogaster. In the last species, N. viridula, a single phenotypic mutant could be detected having a patchy white body coloration with wild type eye coloration. Genotyping revealed a single amino acid deletion at the target site, suggesting the creation of a hypomorphic allele. To conclude, the protocols provided in this work can contribute to the genetic study of predatory mites used in biological control, as well as hemipteran pests.
Additional Links: PMID-39615800
Publisher:
PubMed:
Citation:
show bibtex listing
hide bibtex listing
@article {pmid39615800,
year = {2025},
author = {Mocchetti, A and De Rouck, S and Naessens, S and Dermauw, W and Van Leeuwen, T},
title = {SYNCAS based CRISPR-Cas9 gene editing in predatory mites, whiteflies and stinkbugs.},
journal = {Insect biochemistry and molecular biology},
volume = {177},
number = {},
pages = {104232},
doi = {10.1016/j.ibmb.2024.104232},
pmid = {39615800},
issn = {1879-0240},
mesh = {Animals ; *CRISPR-Cas Systems ; *Gene Editing/methods ; *Hemiptera/genetics ; *Mites/genetics ; },
abstract = {Despite the establishment of CRISPR-Cas9 gene editing protocols in a wide range of organisms, genetic engineering is still challenging for many organisms due to constraints including lethality of embryo injection, difficulties in egg/embryo collection or viviparous lifestyles. Recently, an efficient CRISPR-Cas9 method, termed SYNCAS, was developed to genetically modify spider mites and thrips species. The method is based on maternal injection of formulated CRISPR-Cas9 using saponin and BAPC. Here, we investigate whether the method can be used to perform gene editing in other arthropods such as the beneficial predatory mites Amblyseius swirskii and Phytoseiulus persimilis, and the pests Bemisia tabaci and Nezara viridula. For the predatory mites, Antp and SLC25A38 were used as target genes, while the ortholog of the Drosophila melanogaster ABCG transporter white was targeted in B. tabaci and N. viridula. All species were successfully edited with the highest efficiencies (up to 39%) being obtained for B. tabaci. For A. swirskii and P. persimilis no clear phenotypes could be observed, even though SLC25A38 was successfully knocked-out. The lack of a color phenotype in SLC25A38 mutants was confirmed in the spider mite Tetranychus urticae. Disruption of the target gene Antp is likely lethal in predatory mites, as no true null mutants could be recovered. For B. tabaci, KO of white resulted in orange eyes which diverges from the phenotype seen in white mutants of D. melanogaster. In the last species, N. viridula, a single phenotypic mutant could be detected having a patchy white body coloration with wild type eye coloration. Genotyping revealed a single amino acid deletion at the target site, suggesting the creation of a hypomorphic allele. To conclude, the protocols provided in this work can contribute to the genetic study of predatory mites used in biological control, as well as hemipteran pests.},
}
MeSH Terms:
show MeSH Terms
hide MeSH Terms
Animals
*CRISPR-Cas Systems
*Gene Editing/methods
*Hemiptera/genetics
*Mites/genetics
RevDate: 2025-02-07
CmpDate: 2025-02-08
Loss of MEF2C function by enhancer mutation leads to neuronal mitochondria dysfunction and motor deficits in mice.
Molecular neurodegeneration, 20(1):16.
BACKGROUND: Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder characterized by the loss of both upper and lower motor neurons, leading to progressive paralysis. Both genetic alterations and epigenetic modifications contribute to neuronal dysfunction in the pathogenesis of ALS. However, the mechanism behind genetic mutations in the non-coding region of genes that affect epigenetic modifications remains unclear.
METHODS: Convolutional neural network was used to identify an ALS-associated SNP located in the intronic region of MEF2C (rs304152), residing in a putative enhancer element. To examine the alteration of MEF2C transcription by the SNP, we generated HEK293T cells carrying the major or minor allele by CRISPR-Cas9. To verify the role of MEF2C-knockdown (MEF2C-KD) in mice, we developed AAV expressing shRNA for MEF2C based on AAV-U6 promoter vector. Neuropathological alterations of MEF2C-KD mice with mitochondrial dysfunction and motor neuronal damage were observed by confocal microscopy and transmission electron microscope (TEM). Behavioral changes of mice were examined through longitudinal study by tail suspension, inverted grid test and automated gait analysis.
RESULTS: Here, we show that enhancer mutation of MEF2C reduces own gene expression and consequently impairs mitochondrial function in motor neurons. MEF2C localizes and binds to the mitochondria DNA, and directly modulates mitochondria-encoded gene expression. CRISPR/Cas-9-induced mutation of the MEF2C enhancer decreases expression of mitochondria-encoded genes. Moreover, MEF2C mutant cells show reduction of mitochondrial membrane potential, ATP level but elevation of oxidative stress. MEF2C deficiency in the upper and lower motor neurons of mice impairs mitochondria-encoded genes, and leads to mitochondrial metabolic disruption and progressive motor behavioral deficits.
CONCLUSIONS: Together, MEF2C dysregulation by the enhancer mutation leads to mitochondrial dysfunction and oxidative stress, which are prevalent features in motor neuronal damage and ALS pathogenesis. This genetic and epigenetic crosstalk mechanism provides insights for advancing our understanding of motor neuron disease and developing effective treatments.
Additional Links: PMID-39920775
PubMed:
Citation:
show bibtex listing
hide bibtex listing
@article {pmid39920775,
year = {2025},
author = {Yousefian-Jazi, A and Kim, S and Chu, J and Choi, SH and Nguyen, PTT and Park, U and Kim, MG and Hwang, H and Lee, K and Kim, Y and Hyeon, SJ and Rhim, H and Ryu, HL and Lim, G and Stein, TD and Lim, K and Ryu, H and Lee, J},
title = {Loss of MEF2C function by enhancer mutation leads to neuronal mitochondria dysfunction and motor deficits in mice.},
journal = {Molecular neurodegeneration},
volume = {20},
number = {1},
pages = {16},
pmid = {39920775},
issn = {1750-1326},
support = {2022R1A2C3013138//National Research Foundation/ ; HU23C0217//Korea Dementia Research Project Grant/ ; 2E30954//KIST Grant/ ; R01NS109537//NIH R01/ ; },
mesh = {Animals ; *MEF2 Transcription Factors/metabolism/genetics ; Mice ; *Mitochondria/metabolism ; Humans ; *Motor Neurons/metabolism/pathology ; Amyotrophic Lateral Sclerosis/genetics/metabolism/pathology ; Disease Models, Animal ; Mutation/genetics ; Enhancer Elements, Genetic/genetics ; HEK293 Cells ; },
abstract = {BACKGROUND: Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder characterized by the loss of both upper and lower motor neurons, leading to progressive paralysis. Both genetic alterations and epigenetic modifications contribute to neuronal dysfunction in the pathogenesis of ALS. However, the mechanism behind genetic mutations in the non-coding region of genes that affect epigenetic modifications remains unclear.
METHODS: Convolutional neural network was used to identify an ALS-associated SNP located in the intronic region of MEF2C (rs304152), residing in a putative enhancer element. To examine the alteration of MEF2C transcription by the SNP, we generated HEK293T cells carrying the major or minor allele by CRISPR-Cas9. To verify the role of MEF2C-knockdown (MEF2C-KD) in mice, we developed AAV expressing shRNA for MEF2C based on AAV-U6 promoter vector. Neuropathological alterations of MEF2C-KD mice with mitochondrial dysfunction and motor neuronal damage were observed by confocal microscopy and transmission electron microscope (TEM). Behavioral changes of mice were examined through longitudinal study by tail suspension, inverted grid test and automated gait analysis.
RESULTS: Here, we show that enhancer mutation of MEF2C reduces own gene expression and consequently impairs mitochondrial function in motor neurons. MEF2C localizes and binds to the mitochondria DNA, and directly modulates mitochondria-encoded gene expression. CRISPR/Cas-9-induced mutation of the MEF2C enhancer decreases expression of mitochondria-encoded genes. Moreover, MEF2C mutant cells show reduction of mitochondrial membrane potential, ATP level but elevation of oxidative stress. MEF2C deficiency in the upper and lower motor neurons of mice impairs mitochondria-encoded genes, and leads to mitochondrial metabolic disruption and progressive motor behavioral deficits.
CONCLUSIONS: Together, MEF2C dysregulation by the enhancer mutation leads to mitochondrial dysfunction and oxidative stress, which are prevalent features in motor neuronal damage and ALS pathogenesis. This genetic and epigenetic crosstalk mechanism provides insights for advancing our understanding of motor neuron disease and developing effective treatments.},
}
MeSH Terms:
show MeSH Terms
hide MeSH Terms
Animals
*MEF2 Transcription Factors/metabolism/genetics
Mice
*Mitochondria/metabolism
Humans
*Motor Neurons/metabolism/pathology
Amyotrophic Lateral Sclerosis/genetics/metabolism/pathology
Disease Models, Animal
Mutation/genetics
Enhancer Elements, Genetic/genetics
HEK293 Cells
RevDate: 2025-02-07
CmpDate: 2025-02-08
Synergistic strategies for glioblastoma treatment: CRISPR-based multigene editing combined with immune checkpoint blockade.
Journal of nanobiotechnology, 23(1):94.
Glioblastoma (GBM) is a primary brain tumor known for its high levels of aggressiveness and resistance to current treatments such as radiotherapy and chemotherapy. As a result, there is a pressing need for innovative therapeutic approaches to combat GBM. Thus, we have developed an engineered multifunctional extracellular vesicle (EV) delivery system that offers an "all-in-one" strategy for GBM therapy. Our approach involved the use of genetic engineering to the long-lasting production of PD-1 and the brain-specific peptide angiopep-2 on the surface of EVs. These modified EVs were then utilized to rejuvenate exhausted CD8[+] T cells blocking PD-L1, resulting in significant therapeutic benefits for GBM treatment. Furthermore, the EVs contained Cas9 protein and sgRNA for precise and minimally invasive gene therapy, which addressing the key barriers associated with in vivo CRISPR‒Cas9 gene editing treatment. The multigene editing of EVs resulted in efficient intratumor multisite gene editing (PLK1: 58.6%, VEGF: 52.7%), leading to the successful apoptosis of tumor cells in vivo and demonstrating an antiangiogenic effect. This research introduces a promising universal platform for combining immune checkpoint blockade therapy with gene editing treatment.
Additional Links: PMID-39920725
PubMed:
Citation:
show bibtex listing
hide bibtex listing
@article {pmid39920725,
year = {2025},
author = {Liu, X and Liu, X and Luo, X and Zhu, M and Liu, N and Li, J and Zhang, Q and Zou, C and Wu, Y and Cao, Z and Ma, S and Wang, W and Yang, G and Gu, J and Liu, W and Li, M and Yin, A and He, Y and Lin, W},
title = {Synergistic strategies for glioblastoma treatment: CRISPR-based multigene editing combined with immune checkpoint blockade.},
journal = {Journal of nanobiotechnology},
volume = {23},
number = {1},
pages = {94},
pmid = {39920725},
issn = {1477-3155},
support = {82003220//the National Natural Science Foundation of China/ ; },
mesh = {*Glioblastoma/therapy/genetics ; *Gene Editing/methods ; *Immune Checkpoint Inhibitors/pharmacology/therapeutic use ; *CRISPR-Cas Systems ; Animals ; Humans ; *Brain Neoplasms/therapy/genetics ; Cell Line, Tumor ; Mice ; *Programmed Cell Death 1 Receptor ; Extracellular Vesicles ; Genetic Therapy/methods ; B7-H1 Antigen/genetics ; CD8-Positive T-Lymphocytes/immunology ; },
abstract = {Glioblastoma (GBM) is a primary brain tumor known for its high levels of aggressiveness and resistance to current treatments such as radiotherapy and chemotherapy. As a result, there is a pressing need for innovative therapeutic approaches to combat GBM. Thus, we have developed an engineered multifunctional extracellular vesicle (EV) delivery system that offers an "all-in-one" strategy for GBM therapy. Our approach involved the use of genetic engineering to the long-lasting production of PD-1 and the brain-specific peptide angiopep-2 on the surface of EVs. These modified EVs were then utilized to rejuvenate exhausted CD8[+] T cells blocking PD-L1, resulting in significant therapeutic benefits for GBM treatment. Furthermore, the EVs contained Cas9 protein and sgRNA for precise and minimally invasive gene therapy, which addressing the key barriers associated with in vivo CRISPR‒Cas9 gene editing treatment. The multigene editing of EVs resulted in efficient intratumor multisite gene editing (PLK1: 58.6%, VEGF: 52.7%), leading to the successful apoptosis of tumor cells in vivo and demonstrating an antiangiogenic effect. This research introduces a promising universal platform for combining immune checkpoint blockade therapy with gene editing treatment.},
}
MeSH Terms:
show MeSH Terms
hide MeSH Terms
*Glioblastoma/therapy/genetics
*Gene Editing/methods
*Immune Checkpoint Inhibitors/pharmacology/therapeutic use
*CRISPR-Cas Systems
Animals
Humans
*Brain Neoplasms/therapy/genetics
Cell Line, Tumor
Mice
*Programmed Cell Death 1 Receptor
Extracellular Vesicles
Genetic Therapy/methods
B7-H1 Antigen/genetics
CD8-Positive T-Lymphocytes/immunology
RevDate: 2025-02-07
CmpDate: 2025-02-07
Enhanced genome editing with a Streptococcus equinus Cas9.
Communications biology, 8(1):196.
A large number of SpCas9 orthologs has been computationally identified, but their genome editing potential remains largely unknown. In this study, a GFP-activation assay was used to screen a panel of 18 SpCas9 orthologs, ten of which demonstrated activity in human cells. Notably, these orthologs had a preference for purine-rich PAM sequences. Four of the tested orthologs displayed enhanced specificity compared to SpCas9. Of particular interest is SeqCas9, which recognizes a simple NNG PAM and displays activity and specificity comparable to SpCas9-HF1. In addition, SeqCas9 exhibits superior base editing efficiency compared to SpCas9-NG and SpCas9-NRRH at multiple endogenous loci. This research sheds light on the diversity of SpCas9 orthologs and their potential for specific and efficient genome editing, especially in cases involving base editing.
Additional Links: PMID-39920233
PubMed:
Citation:
show bibtex listing
hide bibtex listing
@article {pmid39920233,
year = {2025},
author = {Liu, J and Wang, Y and Wei, J and Wang, S and Li, M and Huang, Z and Zhang, S and Liu, H and Huang, J and Wang, Y},
title = {Enhanced genome editing with a Streptococcus equinus Cas9.},
journal = {Communications biology},
volume = {8},
number = {1},
pages = {196},
pmid = {39920233},
issn = {2399-3642},
mesh = {*Gene Editing/methods ; Humans ; *CRISPR-Associated Protein 9/genetics/metabolism ; *CRISPR-Cas Systems ; HEK293 Cells ; Streptococcus/genetics ; },
abstract = {A large number of SpCas9 orthologs has been computationally identified, but their genome editing potential remains largely unknown. In this study, a GFP-activation assay was used to screen a panel of 18 SpCas9 orthologs, ten of which demonstrated activity in human cells. Notably, these orthologs had a preference for purine-rich PAM sequences. Four of the tested orthologs displayed enhanced specificity compared to SpCas9. Of particular interest is SeqCas9, which recognizes a simple NNG PAM and displays activity and specificity comparable to SpCas9-HF1. In addition, SeqCas9 exhibits superior base editing efficiency compared to SpCas9-NG and SpCas9-NRRH at multiple endogenous loci. This research sheds light on the diversity of SpCas9 orthologs and their potential for specific and efficient genome editing, especially in cases involving base editing.},
}
MeSH Terms:
show MeSH Terms
hide MeSH Terms
*Gene Editing/methods
Humans
*CRISPR-Associated Protein 9/genetics/metabolism
*CRISPR-Cas Systems
HEK293 Cells
Streptococcus/genetics
RevDate: 2025-02-08
CmpDate: 2025-02-08
Droplet-based functional CRISPR screening of cell-cell interactions by SPEAC-seq.
Nature protocols, 20(2):440-461.
Cell-cell interactions are essential for the function and contextual regulation of biological tissues. We present a platform for high-throughput microfluidics-supported genetic screening of functional regulators of cell-cell interactions. Systematic perturbation of encapsulated associated cells followed by sequencing (SPEAC-seq) combines genome-wide CRISPR libraries, cell coculture in droplets and microfluidic droplet sorting based on functional read-outs determined by fluorescent reporter circuits to enable the unbiased discovery of interaction regulators. This technique overcomes limitations of traditional methods for characterization of cell-cell communication, which require a priori knowledge of cellular interactions, are highly engineered and lack functional read-outs. As an example of this technique, we describe the investigation of neuroinflammatory intercellular communication between microglia and astrocytes, using genome-wide CRISPR-Cas9 inactivation libraries and fluorescent reporters of NF-κB activation. This approach enabled the discovery of thousands of microglial regulators of astrocyte NF-κB activation important for the control of central nervous system inflammation. Importantly, SPEAC-seq can be adapted to different cell types, screening modalities, cell functions and physiological contexts, only limited by the ability to fluorescently report cell functions and by droplet cultivation conditions. Performing genome-wide screening takes less than 2 weeks and requires microfluidics capabilities. Thus, SPEAC-seq enables the large-scale investigation of cell-cell interactions.
Additional Links: PMID-39327538
PubMed:
Citation:
show bibtex listing
hide bibtex listing
@article {pmid39327538,
year = {2025},
author = {Faust Akl, C and Linnerbauer, M and Li, Z and Lee, HG and Clark, IC and Wheeler, MA and Quintana, FJ},
title = {Droplet-based functional CRISPR screening of cell-cell interactions by SPEAC-seq.},
journal = {Nature protocols},
volume = {20},
number = {2},
pages = {440-461},
pmid = {39327538},
issn = {1750-2799},
support = {R01 MH130458/MH/NIMH NIH HHS/United States ; R00 NS114111/NS/NINDS NIH HHS/United States ; R01 MH132632/MH/NIMH NIH HHS/United States ; R01 DA061199/DA/NIDA NIH HHS/United States ; L30 NS139299/NS/NINDS NIH HHS/United States ; },
mesh = {*Cell Communication/genetics ; *Astrocytes/metabolism ; *Microglia/metabolism/cytology ; *CRISPR-Cas Systems ; Animals ; Mice ; NF-kappa B/metabolism ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Humans ; Microfluidics/methods ; Coculture Techniques/methods ; },
abstract = {Cell-cell interactions are essential for the function and contextual regulation of biological tissues. We present a platform for high-throughput microfluidics-supported genetic screening of functional regulators of cell-cell interactions. Systematic perturbation of encapsulated associated cells followed by sequencing (SPEAC-seq) combines genome-wide CRISPR libraries, cell coculture in droplets and microfluidic droplet sorting based on functional read-outs determined by fluorescent reporter circuits to enable the unbiased discovery of interaction regulators. This technique overcomes limitations of traditional methods for characterization of cell-cell communication, which require a priori knowledge of cellular interactions, are highly engineered and lack functional read-outs. As an example of this technique, we describe the investigation of neuroinflammatory intercellular communication between microglia and astrocytes, using genome-wide CRISPR-Cas9 inactivation libraries and fluorescent reporters of NF-κB activation. This approach enabled the discovery of thousands of microglial regulators of astrocyte NF-κB activation important for the control of central nervous system inflammation. Importantly, SPEAC-seq can be adapted to different cell types, screening modalities, cell functions and physiological contexts, only limited by the ability to fluorescently report cell functions and by droplet cultivation conditions. Performing genome-wide screening takes less than 2 weeks and requires microfluidics capabilities. Thus, SPEAC-seq enables the large-scale investigation of cell-cell interactions.},
}
MeSH Terms:
show MeSH Terms
hide MeSH Terms
*Cell Communication/genetics
*Astrocytes/metabolism
*Microglia/metabolism/cytology
*CRISPR-Cas Systems
Animals
Mice
NF-kappa B/metabolism
Clustered Regularly Interspaced Short Palindromic Repeats/genetics
Humans
Microfluidics/methods
Coculture Techniques/methods
RevDate: 2025-02-07
CmpDate: 2025-02-07
Two is better than one: Advancing gene therapy protocols for enhanced safety and efficacy.
Cell stem cell, 32(2):179-180.
In this issue of Cell Stem Cell, two complementary studies from Zeng et al.[1] and Demirci et al.[2] improve the efficacy of gene therapy for β-hemoglobinopathies using multiplex CRISPR-Cas9 editing. The authors also optimize editing and conditioning protocols to reduce risks associated with current gene therapy procedures.
Additional Links: PMID-39919720
Publisher:
PubMed:
Citation:
show bibtex listing
hide bibtex listing
@article {pmid39919720,
year = {2025},
author = {Hardouin, G and Miccio, A},
title = {Two is better than one: Advancing gene therapy protocols for enhanced safety and efficacy.},
journal = {Cell stem cell},
volume = {32},
number = {2},
pages = {179-180},
doi = {10.1016/j.stem.2024.12.009},
pmid = {39919720},
issn = {1875-9777},
mesh = {*Genetic Therapy/methods ; Humans ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; },
abstract = {In this issue of Cell Stem Cell, two complementary studies from Zeng et al.[1] and Demirci et al.[2] improve the efficacy of gene therapy for β-hemoglobinopathies using multiplex CRISPR-Cas9 editing. The authors also optimize editing and conditioning protocols to reduce risks associated with current gene therapy procedures.},
}
MeSH Terms:
show MeSH Terms
hide MeSH Terms
*Genetic Therapy/methods
Humans
*Gene Editing/methods
*CRISPR-Cas Systems/genetics
RevDate: 2025-02-07
Blood cancer therapy with synthetic receptors and CRISPR technology.
Leukemia research, 150:107646 pii:S0145-2126(25)00006-2 [Epub ahead of print].
Chimeric antigen receptor (CAR)-T and -NK cells showed great success in treating hematological malignancies, including leukemia, lymphoma, and myeloma. CRISPR technology and other synthetic receptors (GPCR and synNotch) have helped to address some of the limitations and challenges associated with CAR-based therapies. Herein, this review aims to discuss how CAR can be integrated with other synthetic receptors and various CRISPR/Cas tools for blood cancer therapy. CAR-expressing cells equipped with other synthetic receptors can conditionally execute tumoricidal functions, prevent tumor escape from immune surveillance, and minimize non-tumor off-target toxicity. We also discussed how various CRISPR-Cas tools can be harnessed to enhance CAR cells functionality and persistence. The advances, pitfalls, and future perspectives for these synthetic receptors and CRISPR technology in blood cancer therapy are comprehensively discussed.
Additional Links: PMID-39919536
Publisher:
PubMed:
Citation:
show bibtex listing
hide bibtex listing
@article {pmid39919536,
year = {2025},
author = {Zhang, H and Zhong, M and Zhang, J and Chen, C},
title = {Blood cancer therapy with synthetic receptors and CRISPR technology.},
journal = {Leukemia research},
volume = {150},
number = {},
pages = {107646},
doi = {10.1016/j.leukres.2025.107646},
pmid = {39919536},
issn = {1873-5835},
abstract = {Chimeric antigen receptor (CAR)-T and -NK cells showed great success in treating hematological malignancies, including leukemia, lymphoma, and myeloma. CRISPR technology and other synthetic receptors (GPCR and synNotch) have helped to address some of the limitations and challenges associated with CAR-based therapies. Herein, this review aims to discuss how CAR can be integrated with other synthetic receptors and various CRISPR/Cas tools for blood cancer therapy. CAR-expressing cells equipped with other synthetic receptors can conditionally execute tumoricidal functions, prevent tumor escape from immune surveillance, and minimize non-tumor off-target toxicity. We also discussed how various CRISPR-Cas tools can be harnessed to enhance CAR cells functionality and persistence. The advances, pitfalls, and future perspectives for these synthetic receptors and CRISPR technology in blood cancer therapy are comprehensively discussed.},
}
RevDate: 2025-02-06
CmpDate: 2025-02-06
A new 'mini-CRISPR' flexes its editing power in monkey muscles.
Science (New York, N.Y.), 387(6734):570.
The downsized DNA-slicing machinery may reach more tissues to take aim at more diseases.
Additional Links: PMID-39913574
Publisher:
PubMed:
Citation:
show bibtex listing
hide bibtex listing
@article {pmid39913574,
year = {2025},
author = {Couzin-Frankel, J},
title = {A new 'mini-CRISPR' flexes its editing power in monkey muscles.},
journal = {Science (New York, N.Y.)},
volume = {387},
number = {6734},
pages = {570},
doi = {10.1126/science.adw4916},
pmid = {39913574},
issn = {1095-9203},
mesh = {Animals ; *Gene Editing ; *CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Muscle, Skeletal ; CRISPR-Associated Protein 9 ; Humans ; },
abstract = {The downsized DNA-slicing machinery may reach more tissues to take aim at more diseases.},
}
MeSH Terms:
show MeSH Terms
hide MeSH Terms
Animals
*Gene Editing
*CRISPR-Cas Systems
Clustered Regularly Interspaced Short Palindromic Repeats
Muscle, Skeletal
CRISPR-Associated Protein 9
Humans
RevDate: 2025-02-06
CmpDate: 2025-02-06
LRRC56 deletion causes primary ciliary dyskinesia in mice characterized by dynein arms defects.
Biology open, 14(2):.
Leucine Rich Repeat Containing protein 56 (LRRC56), also known as DNAAF12, is a member of the LRRC superfamily, whose dysfunction is associated with mucociliary clearance and laterality defects in humans. Here, we generated LRRC56-knockout mice using the CRISPR/Cas9 nuclease system to specifically target exons 4-5 of the LRRC56 gene. We observed that homozygous LRRC56 gene deletion is definitely deleterious, as 27.8% of LRRC56-/- mice died before adulthood. Among the surviving LRRC56-/- mice, the most prominent phenotypes included hydrocephalus, situs inversus, male infertility, and bronchiectasis. Transmission electron microscopy revealed defects in dynein arms of cilia and disorganized axonemal structure in flagella. Immunofluorescence analysis similarly revealed the absence of inner and outer dynein arm markers DNALI1 and DNAI2 in the cilia. Heterozygous LRRC56+/- mice developed normally, without exhibiting any symptoms of primary ciliary dyskinesia. In conclusion, the knockout of the LRRC56 gene in mice leads to a range of conditions consistent with primary ciliary dyskinesia. The absence of DNALI1 and DNAI2 signaling in knockout mouse cilia supports the critical role of the LRRC56 gene in dynein arm assembly.
Additional Links: PMID-39912490
Publisher:
PubMed:
Citation:
show bibtex listing
hide bibtex listing
@article {pmid39912490,
year = {2025},
author = {Wu, R and Li, H and Wu, P and Yang, Q and Wan, X and Wu, Y},
title = {LRRC56 deletion causes primary ciliary dyskinesia in mice characterized by dynein arms defects.},
journal = {Biology open},
volume = {14},
number = {2},
pages = {},
doi = {10.1242/bio.061846},
pmid = {39912490},
issn = {2046-6390},
support = {82002238//National Natural Science Foundation/ ; //Central South University Third Xiangya Hospital/ ; },
mesh = {Animals ; Mice ; *Mice, Knockout ; *Cilia/metabolism ; *Dyneins/genetics/metabolism ; *Disease Models, Animal ; *Phenotype ; Gene Deletion ; CRISPR-Cas Systems ; Male ; Ciliary Motility Disorders/genetics ; Kartagener Syndrome/genetics ; },
abstract = {Leucine Rich Repeat Containing protein 56 (LRRC56), also known as DNAAF12, is a member of the LRRC superfamily, whose dysfunction is associated with mucociliary clearance and laterality defects in humans. Here, we generated LRRC56-knockout mice using the CRISPR/Cas9 nuclease system to specifically target exons 4-5 of the LRRC56 gene. We observed that homozygous LRRC56 gene deletion is definitely deleterious, as 27.8% of LRRC56-/- mice died before adulthood. Among the surviving LRRC56-/- mice, the most prominent phenotypes included hydrocephalus, situs inversus, male infertility, and bronchiectasis. Transmission electron microscopy revealed defects in dynein arms of cilia and disorganized axonemal structure in flagella. Immunofluorescence analysis similarly revealed the absence of inner and outer dynein arm markers DNALI1 and DNAI2 in the cilia. Heterozygous LRRC56+/- mice developed normally, without exhibiting any symptoms of primary ciliary dyskinesia. In conclusion, the knockout of the LRRC56 gene in mice leads to a range of conditions consistent with primary ciliary dyskinesia. The absence of DNALI1 and DNAI2 signaling in knockout mouse cilia supports the critical role of the LRRC56 gene in dynein arm assembly.},
}
MeSH Terms:
show MeSH Terms
hide MeSH Terms
Animals
Mice
*Mice, Knockout
*Cilia/metabolism
*Dyneins/genetics/metabolism
*Disease Models, Animal
*Phenotype
Gene Deletion
CRISPR-Cas Systems
Male
Ciliary Motility Disorders/genetics
Kartagener Syndrome/genetics
RevDate: 2025-02-07
CmpDate: 2025-02-07
Gene editing without ex vivo culture evades genotoxicity in human hematopoietic stem cells.
Cell stem cell, 32(2):191-208.e11.
Gene editing the BCL11A erythroid enhancer is a validated approach to fetal hemoglobin (HbF) induction for β-hemoglobinopathy therapy, though heterogeneity in edit allele distribution and HbF response may impact its safety and efficacy. Here, we compare combined CRISPR-Cas9 editing of the BCL11A +58 and +55 enhancers with leading gene modification approaches under clinical investigation. Dual targeting of the BCL11A +58 and +55 enhancers with 3xNLS-SpCas9 and two single guide RNAs (sgRNAs) resulted in superior HbF induction, including in sickle cell disease (SCD) patient xenografts, attributable to simultaneous disruption of core half E-box/GATA motifs at both enhancers. Unintended on-target outcomes of double-strand break (DSB) repair in hematopoietic stem and progenitor cells (HSPCs), such as long deletions and centromere-distal chromosome fragment loss, are a byproduct of cellular proliferation stimulated by ex vivo culture. Editing quiescent HSPCs bypasses long deletion and micronuclei formation and preserves efficient on-target editing and engraftment function.
Additional Links: PMID-39672163
Publisher:
PubMed:
Citation:
show bibtex listing
hide bibtex listing
@article {pmid39672163,
year = {2025},
author = {Zeng, J and Nguyen, MA and Liu, P and da Silva, LF and Levesque, S and Lin, LY and Justus, DG and Petri, K and Clement, K and Porter, SN and Verma, A and Neri, NR and Rosanwo, T and Ciuculescu, MF and Abriss, D and Mintzer, E and Maitland, SA and Demirci, S and Cha, HJ and Orkin, SH and Tisdale, JF and Williams, DA and Zhu, LJ and Pruett-Miller, SM and Pinello, L and Joung, JK and Pattanayak, V and Manis, JP and Armant, M and Pellin, D and Brendel, C and Wolfe, SA and Bauer, DE},
title = {Gene editing without ex vivo culture evades genotoxicity in human hematopoietic stem cells.},
journal = {Cell stem cell},
volume = {32},
number = {2},
pages = {191-208.e11},
doi = {10.1016/j.stem.2024.11.001},
pmid = {39672163},
issn = {1875-9777},
support = {R01 HL150669/HL/NHLBI NIH HHS/United States ; RM1 HG009490/HG/NHGRI NIH HHS/United States ; },
mesh = {Humans ; *Hematopoietic Stem Cells/metabolism ; *Gene Editing ; Animals ; CRISPR-Cas Systems/genetics ; Anemia, Sickle Cell/genetics/therapy/pathology ; Mice ; Fetal Hemoglobin/genetics/metabolism ; Hematopoietic Stem Cell Transplantation ; Enhancer Elements, Genetic/genetics ; Repressor Proteins ; },
abstract = {Gene editing the BCL11A erythroid enhancer is a validated approach to fetal hemoglobin (HbF) induction for β-hemoglobinopathy therapy, though heterogeneity in edit allele distribution and HbF response may impact its safety and efficacy. Here, we compare combined CRISPR-Cas9 editing of the BCL11A +58 and +55 enhancers with leading gene modification approaches under clinical investigation. Dual targeting of the BCL11A +58 and +55 enhancers with 3xNLS-SpCas9 and two single guide RNAs (sgRNAs) resulted in superior HbF induction, including in sickle cell disease (SCD) patient xenografts, attributable to simultaneous disruption of core half E-box/GATA motifs at both enhancers. Unintended on-target outcomes of double-strand break (DSB) repair in hematopoietic stem and progenitor cells (HSPCs), such as long deletions and centromere-distal chromosome fragment loss, are a byproduct of cellular proliferation stimulated by ex vivo culture. Editing quiescent HSPCs bypasses long deletion and micronuclei formation and preserves efficient on-target editing and engraftment function.},
}
MeSH Terms:
show MeSH Terms
hide MeSH Terms
Humans
*Hematopoietic Stem Cells/metabolism
*Gene Editing
Animals
CRISPR-Cas Systems/genetics
Anemia, Sickle Cell/genetics/therapy/pathology
Mice
Fetal Hemoglobin/genetics/metabolism
Hematopoietic Stem Cell Transplantation
Enhancer Elements, Genetic/genetics
Repressor Proteins
RevDate: 2025-02-06
CmpDate: 2025-02-06
3' UTR-truncated HMGA2 promotes erythroblasts production from human embryonic stem cells.
Stem cells translational medicine, 14(1):.
Cultured red blood cells represent an alternative resource for blood transfusions. However, important issues such as low yields and high costs remain. Recently, gene editing of hematopoietic stem cells has been conducted to induce erythroid differentiation in vitro for producing sufficient RBCs to meet the imbalance in blood supply and demand. The differentiation and expansion of hematopoietic stem and progenitor cells are regulated by transcription factors, such as high mobility group AT-hook 2 (HMGA2). In this study, we utilized CRISPR/Cas9 to establish a doxycycline-inducible HMGA2-expressing human embryonic stem cell (hESC) line. In a defined erythroid differentiation system, HMGA2 prolonged erythroid differentiation in vitro, enabling extensive expansion of human erythroblasts. The erythroblasts derived from the HMGA2-expressing hESC line are rich in polychromatic and orthochromatic erythroblasts expressing mostly α- and γ-globin and have the capacity to differentiate into RBCs. Our findings highlight the potential of combining hematopoietic transcription factors with genome editing techniques to enhance RBC production.
Additional Links: PMID-39912395
Publisher:
PubMed:
Citation:
show bibtex listing
hide bibtex listing
@article {pmid39912395,
year = {2025},
author = {Cui, T and Wang, X and Zang, R and Zhao, L and Yan, H and Li, X and Xu, Z and Wang, H and Zhou, J and Liu, Y and Yue, W and Pei, X and Xi, J},
title = {3' UTR-truncated HMGA2 promotes erythroblasts production from human embryonic stem cells.},
journal = {Stem cells translational medicine},
volume = {14},
number = {1},
pages = {},
doi = {10.1093/stcltm/szaf001},
pmid = {39912395},
issn = {2157-6580},
support = {82200690//National Natural Science Foundation of China/ ; },
mesh = {Humans ; *Erythroblasts/metabolism/cytology ; *Human Embryonic Stem Cells/metabolism/cytology ; *HMGA2 Protein/metabolism/genetics ; *Cell Differentiation ; *3' Untranslated Regions ; CRISPR-Cas Systems ; Gene Editing/methods ; Cell Line ; Erythropoiesis ; },
abstract = {Cultured red blood cells represent an alternative resource for blood transfusions. However, important issues such as low yields and high costs remain. Recently, gene editing of hematopoietic stem cells has been conducted to induce erythroid differentiation in vitro for producing sufficient RBCs to meet the imbalance in blood supply and demand. The differentiation and expansion of hematopoietic stem and progenitor cells are regulated by transcription factors, such as high mobility group AT-hook 2 (HMGA2). In this study, we utilized CRISPR/Cas9 to establish a doxycycline-inducible HMGA2-expressing human embryonic stem cell (hESC) line. In a defined erythroid differentiation system, HMGA2 prolonged erythroid differentiation in vitro, enabling extensive expansion of human erythroblasts. The erythroblasts derived from the HMGA2-expressing hESC line are rich in polychromatic and orthochromatic erythroblasts expressing mostly α- and γ-globin and have the capacity to differentiate into RBCs. Our findings highlight the potential of combining hematopoietic transcription factors with genome editing techniques to enhance RBC production.},
}
MeSH Terms:
show MeSH Terms
hide MeSH Terms
Humans
*Erythroblasts/metabolism/cytology
*Human Embryonic Stem Cells/metabolism/cytology
*HMGA2 Protein/metabolism/genetics
*Cell Differentiation
*3' Untranslated Regions
CRISPR-Cas Systems
Gene Editing/methods
Cell Line
Erythropoiesis
RevDate: 2025-02-06
CmpDate: 2025-02-06
Identification and characterization of DICER-LIKE genes and their roles in Marchantia polymorpha development and salt stress response.
The Plant journal : for cell and molecular biology, 121(3):e17236.
DICER-LIKE (DCL) proteins play a central role in plant small RNA (sRNA) biogenesis. The genome of the early land plant Marchantia polymorpha encodes four DCL proteins: MpDCL1a, MpDCL1b, MpDCL3, and MpDCL4. While MpDCL1a, MpDCL3 and MpDCL4 show high similarities to their orthologs in Physcomitrium patens and Arabidopsis thaliana, MpDCL1b shares only a limited homology with PpDCL1b, but it is very similar, in terms of functional domains, to orthologs in other moss and fern species. We generated Mpdcl[ge] mutant lines for all MpDCL genes with the CRISPR/Cas9 system and conducted phenotypic analyses under control, salt stress, and phytohormone treatments to uncover specific MpDCL functions. The mutants displayed severe developmental aberrations, altered responses to salt and phytohormones, and disturbed sexual organ development. By combining mRNA and sRNA analyses, we demonstrate that MpDCLs and their associated sRNAs play pivotal roles in regulating development, abiotic stress tolerance and phytohormone response in M. polymorpha. We identified MpDCL1a in microRNA biogenesis, MpDCL4 in trans-acting small interfering RNA generation, and MpDCL3 in the regulation of pathogen-related genes. Notably, salt sensitivity in M. polymorpha is dependent on MpDCL1b and Mpdcl1b[ge] mutants display enhanced tolerance and reduced miRNA expression in response to salt stress. We propose that M. polymorpha employs specific mechanisms for regulating MpDCL1b associated miRNAs under high salinity conditions, potentially shared with other species harboring MpDCL1b homologs.
Additional Links: PMID-39910986
Publisher:
PubMed:
Citation:
show bibtex listing
hide bibtex listing
@article {pmid39910986,
year = {2025},
author = {Csicsely, E and Oberender, A and Georgiadou, AS and Alz, J and Kiel, S and Gutsche, N and Zachgo, S and Grünert, J and Klingl, A and Top, O and Frank, W},
title = {Identification and characterization of DICER-LIKE genes and their roles in Marchantia polymorpha development and salt stress response.},
journal = {The Plant journal : for cell and molecular biology},
volume = {121},
number = {3},
pages = {e17236},
doi = {10.1111/tpj.17236},
pmid = {39910986},
issn = {1365-313X},
support = {FR1677/5-1//Deutsche Forschungsgemeinschaft/ ; },
mesh = {*Marchantia/genetics/physiology/growth & development ; *Ribonuclease III/genetics/metabolism ; *Salt Stress/genetics ; *Plant Proteins/genetics/metabolism ; *Gene Expression Regulation, Plant ; Plant Growth Regulators/metabolism ; Mutation ; CRISPR-Cas Systems ; RNA, Plant/genetics ; },
abstract = {DICER-LIKE (DCL) proteins play a central role in plant small RNA (sRNA) biogenesis. The genome of the early land plant Marchantia polymorpha encodes four DCL proteins: MpDCL1a, MpDCL1b, MpDCL3, and MpDCL4. While MpDCL1a, MpDCL3 and MpDCL4 show high similarities to their orthologs in Physcomitrium patens and Arabidopsis thaliana, MpDCL1b shares only a limited homology with PpDCL1b, but it is very similar, in terms of functional domains, to orthologs in other moss and fern species. We generated Mpdcl[ge] mutant lines for all MpDCL genes with the CRISPR/Cas9 system and conducted phenotypic analyses under control, salt stress, and phytohormone treatments to uncover specific MpDCL functions. The mutants displayed severe developmental aberrations, altered responses to salt and phytohormones, and disturbed sexual organ development. By combining mRNA and sRNA analyses, we demonstrate that MpDCLs and their associated sRNAs play pivotal roles in regulating development, abiotic stress tolerance and phytohormone response in M. polymorpha. We identified MpDCL1a in microRNA biogenesis, MpDCL4 in trans-acting small interfering RNA generation, and MpDCL3 in the regulation of pathogen-related genes. Notably, salt sensitivity in M. polymorpha is dependent on MpDCL1b and Mpdcl1b[ge] mutants display enhanced tolerance and reduced miRNA expression in response to salt stress. We propose that M. polymorpha employs specific mechanisms for regulating MpDCL1b associated miRNAs under high salinity conditions, potentially shared with other species harboring MpDCL1b homologs.},
}
MeSH Terms:
show MeSH Terms
hide MeSH Terms
*Marchantia/genetics/physiology/growth & development
*Ribonuclease III/genetics/metabolism
*Salt Stress/genetics
*Plant Proteins/genetics/metabolism
*Gene Expression Regulation, Plant
Plant Growth Regulators/metabolism
Mutation
CRISPR-Cas Systems
RNA, Plant/genetics
RevDate: 2025-02-05
Harnessing crRNA Transformer for Facile and Specific Nucleic Acid Detection.
Analytical chemistry [Epub ahead of print].
CRISPR/Cas systems have emerged as promising tools for nucleic acid detection. However, their practical applications have been limited by poor specificity and the need for additional preprocessing steps. Inspired by the concept of transformers, we found that changing the forms of crRNA with spatial arrangement may endow CRISPR/Cas with an enhanced performance for nucleic acid detection. Specifically, we rationally designed two crRNA transformers─swap crRNA and split crRNA─and found that they direct the CRISPR/Cas system for cis- and trans- cleavage with enhanced specificity and decreased Cas binding affinity and possess both DNA and RNA detection abilities. Based on these findings, our strategy enabled the identification of clinical prostatic cancer in a one-step reaction, with a remarkable sensitivity of 90.0% and specificity of 96.0%. Our study deepens the understanding of CRISPR/Cas systems and introduces a promising strategy for simple nucleic acid detection with enhanced specificity, sensitivity, and functionality in clinical molecular diagnosis.
Additional Links: PMID-39910413
Publisher:
PubMed:
Citation:
show bibtex listing
hide bibtex listing
@article {pmid39910413,
year = {2025},
author = {Wen, M and Zhou, M and Huang, Z and Wang, Y and Wang, M and Ding, Y and Huang, X and Wang, B and Wen, J and Chen, T and Zhang, P and Chen, M and Yang, C and Zhang, XB and Ke, G},
title = {Harnessing crRNA Transformer for Facile and Specific Nucleic Acid Detection.},
journal = {Analytical chemistry},
volume = {},
number = {},
pages = {},
doi = {10.1021/acs.analchem.4c05399},
pmid = {39910413},
issn = {1520-6882},
abstract = {CRISPR/Cas systems have emerged as promising tools for nucleic acid detection. However, their practical applications have been limited by poor specificity and the need for additional preprocessing steps. Inspired by the concept of transformers, we found that changing the forms of crRNA with spatial arrangement may endow CRISPR/Cas with an enhanced performance for nucleic acid detection. Specifically, we rationally designed two crRNA transformers─swap crRNA and split crRNA─and found that they direct the CRISPR/Cas system for cis- and trans- cleavage with enhanced specificity and decreased Cas binding affinity and possess both DNA and RNA detection abilities. Based on these findings, our strategy enabled the identification of clinical prostatic cancer in a one-step reaction, with a remarkable sensitivity of 90.0% and specificity of 96.0%. Our study deepens the understanding of CRISPR/Cas systems and introduces a promising strategy for simple nucleic acid detection with enhanced specificity, sensitivity, and functionality in clinical molecular diagnosis.},
}
RevDate: 2025-02-05
CmpDate: 2025-02-05
Induced protein expression in Leptospira spp. and its application to CRISPR/Cas9 mutant generation.
Scientific reports, 15(1):4334.
Expanding the genetic toolkit for Leptospira spp. is a crucial step toward advancing our understanding of the biology and virulence of these atypical bacteria. Pathogenic Leptospira are responsible for over 1 million human leptospirosis cases annually and significantly impact domestic animals. Bovine leptospirosis causes substantial financial losses due to abortion, stillbirths, and suboptimal reproductive performance. The advent of the CRISPR/Cas9 system has marked a turning point in genetic manipulation, with applications across multiple Leptospira species. However, incorporating controlled protein expression into existing genetic tools could further expand their utility. We developed and demonstrated the functionality of IPTG-inducible heterologous protein expression in Leptospira spp. This system was applied for regulated expression of dead Cas9 (dCas9) to generate knockdown mutants, and Cas9 to produce knockout mutants by inducing double-strand breaks (DSB) into desired targets. IPTG-induced dCas9 expression enabled validation of essential genes and non-coding RNAs. Additionally, IPTG-controlled Cas9 expression combined with a constitutive non-homologous end-joining (NHEJ) system allowed for successful recovery of knockout mutants, even in the absence of IPTG. These newly controlled protein expression systems will advance studies on the basic biology and virulence of Leptospira, as well as facilitate knockout mutant generation for improved veterinary vaccines.
Additional Links: PMID-39910196
PubMed:
Citation:
show bibtex listing
hide bibtex listing
@article {pmid39910196,
year = {2025},
author = {Fernandes, LGV and Nascimento, ALTO and Nally, JE},
title = {Induced protein expression in Leptospira spp. and its application to CRISPR/Cas9 mutant generation.},
journal = {Scientific reports},
volume = {15},
number = {1},
pages = {4334},
pmid = {39910196},
issn = {2045-2322},
mesh = {*CRISPR-Cas Systems ; *Leptospira/genetics/pathogenicity ; Animals ; Leptospirosis/microbiology ; Gene Editing ; Cattle ; Mutation ; Gene Knockout Techniques ; CRISPR-Associated Protein 9/genetics/metabolism ; Bacterial Proteins/genetics/metabolism ; Humans ; },
abstract = {Expanding the genetic toolkit for Leptospira spp. is a crucial step toward advancing our understanding of the biology and virulence of these atypical bacteria. Pathogenic Leptospira are responsible for over 1 million human leptospirosis cases annually and significantly impact domestic animals. Bovine leptospirosis causes substantial financial losses due to abortion, stillbirths, and suboptimal reproductive performance. The advent of the CRISPR/Cas9 system has marked a turning point in genetic manipulation, with applications across multiple Leptospira species. However, incorporating controlled protein expression into existing genetic tools could further expand their utility. We developed and demonstrated the functionality of IPTG-inducible heterologous protein expression in Leptospira spp. This system was applied for regulated expression of dead Cas9 (dCas9) to generate knockdown mutants, and Cas9 to produce knockout mutants by inducing double-strand breaks (DSB) into desired targets. IPTG-induced dCas9 expression enabled validation of essential genes and non-coding RNAs. Additionally, IPTG-controlled Cas9 expression combined with a constitutive non-homologous end-joining (NHEJ) system allowed for successful recovery of knockout mutants, even in the absence of IPTG. These newly controlled protein expression systems will advance studies on the basic biology and virulence of Leptospira, as well as facilitate knockout mutant generation for improved veterinary vaccines.},
}
MeSH Terms:
show MeSH Terms
hide MeSH Terms
*CRISPR-Cas Systems
*Leptospira/genetics/pathogenicity
Animals
Leptospirosis/microbiology
Gene Editing
Cattle
Mutation
Gene Knockout Techniques
CRISPR-Associated Protein 9/genetics/metabolism
Bacterial Proteins/genetics/metabolism
Humans
RevDate: 2025-02-05
CmpDate: 2025-02-05
Whole-genome sequencing of Acinetobacter baumannii clinical isolates from a tertiary hospital in Terengganu, Malaysia (2011-2020), revealed the predominance of the Global Clone 2 lineage.
Microbial genomics, 11(2):.
Carbapenem-resistant Acinetobacter baumannii is recognized by the World Health Organization (WHO) as one of the top priority pathogens. Despite its public health importance, genomic data of clinical isolates from Malaysia remain scarce. In this study, whole-genome sequencing was performed on 126 A. baumannii isolates collected from the main tertiary hospital in the state of Terengganu, Malaysia, over a 10-year period (2011-2020). Antimicrobial susceptibilities determined for 20 antibiotics belonging to 8 classes showed that 77.0% (n=97/126) of the isolates were categorized as multidrug resistant (MDR), with all MDR isolates being carbapenem resistant. Multilocus sequence typing analysis categorized the Terengganu A. baumannii clinical isolates into 34 Pasteur and 44 Oxford sequence types (STs), with ST2Pasteur of the Global Clone 2 lineage identified as the dominant ST (n=76/126; 60.3%). The ST2Pasteur isolates could be subdivided into six Oxford STs with the majority being ST195Oxford (n=35) and ST208Oxford (n=17). Various antimicrobial resistance genes were identified with the bla OXA-23-encoded carbapenemase being the predominant acquired carbapenemase gene (n=90/126; 71.4%). Plasmid-encoded rep genes were identified in nearly all (n=122/126; 96.8%) of the isolates with the majority being Rep_3 family (n=121). Various virulence factors were identified, highlighting the pathogenic nature of this bacterium. Only 14/126 (11.1%) of the isolates were positive for the carriage of CRISPR-Cas arrays with none of the prevalent ST2Pasteur isolates harbouring them. This study provided a genomic snapshot of the A. baumannii isolates obtained from a single tertiary healthcare centre in Malaysia over a 10-year period and showed the predominance of a single closely related ST2Pasteur lineage, indicating the entrenchment of this clone in the hospital.
Additional Links: PMID-39908088
Publisher:
PubMed:
Citation:
show bibtex listing
hide bibtex listing
@article {pmid39908088,
year = {2025},
author = {Din, NS and Mohd Rani, F and Alattraqchi, AG and Ismail, S and A Rahman, NI and Cleary, DW and Clarke, SC and Yeo, CC},
title = {Whole-genome sequencing of Acinetobacter baumannii clinical isolates from a tertiary hospital in Terengganu, Malaysia (2011-2020), revealed the predominance of the Global Clone 2 lineage.},
journal = {Microbial genomics},
volume = {11},
number = {2},
pages = {},
doi = {10.1099/mgen.0.001345},
pmid = {39908088},
issn = {2057-5858},
mesh = {*Acinetobacter baumannii/genetics/drug effects/isolation & purification/classification ; Malaysia/epidemiology ; *Tertiary Care Centers ; Humans ; *Whole Genome Sequencing ; *Drug Resistance, Multiple, Bacterial/genetics ; *Acinetobacter Infections/microbiology/epidemiology ; *Multilocus Sequence Typing ; *Anti-Bacterial Agents/pharmacology ; Microbial Sensitivity Tests ; Genome, Bacterial ; Male ; beta-Lactamases/genetics ; Adult ; Female ; Middle Aged ; Aged ; Bacterial Proteins/genetics ; Young Adult ; Phylogeny ; Adolescent ; Aged, 80 and over ; Child ; },
abstract = {Carbapenem-resistant Acinetobacter baumannii is recognized by the World Health Organization (WHO) as one of the top priority pathogens. Despite its public health importance, genomic data of clinical isolates from Malaysia remain scarce. In this study, whole-genome sequencing was performed on 126 A. baumannii isolates collected from the main tertiary hospital in the state of Terengganu, Malaysia, over a 10-year period (2011-2020). Antimicrobial susceptibilities determined for 20 antibiotics belonging to 8 classes showed that 77.0% (n=97/126) of the isolates were categorized as multidrug resistant (MDR), with all MDR isolates being carbapenem resistant. Multilocus sequence typing analysis categorized the Terengganu A. baumannii clinical isolates into 34 Pasteur and 44 Oxford sequence types (STs), with ST2Pasteur of the Global Clone 2 lineage identified as the dominant ST (n=76/126; 60.3%). The ST2Pasteur isolates could be subdivided into six Oxford STs with the majority being ST195Oxford (n=35) and ST208Oxford (n=17). Various antimicrobial resistance genes were identified with the bla OXA-23-encoded carbapenemase being the predominant acquired carbapenemase gene (n=90/126; 71.4%). Plasmid-encoded rep genes were identified in nearly all (n=122/126; 96.8%) of the isolates with the majority being Rep_3 family (n=121). Various virulence factors were identified, highlighting the pathogenic nature of this bacterium. Only 14/126 (11.1%) of the isolates were positive for the carriage of CRISPR-Cas arrays with none of the prevalent ST2Pasteur isolates harbouring them. This study provided a genomic snapshot of the A. baumannii isolates obtained from a single tertiary healthcare centre in Malaysia over a 10-year period and showed the predominance of a single closely related ST2Pasteur lineage, indicating the entrenchment of this clone in the hospital.},
}
MeSH Terms:
show MeSH Terms
hide MeSH Terms
*Acinetobacter baumannii/genetics/drug effects/isolation & purification/classification
Malaysia/epidemiology
*Tertiary Care Centers
Humans
*Whole Genome Sequencing
*Drug Resistance, Multiple, Bacterial/genetics
*Acinetobacter Infections/microbiology/epidemiology
*Multilocus Sequence Typing
*Anti-Bacterial Agents/pharmacology
Microbial Sensitivity Tests
Genome, Bacterial
Male
beta-Lactamases/genetics
Adult
Female
Middle Aged
Aged
Bacterial Proteins/genetics
Young Adult
Phylogeny
Adolescent
Aged, 80 and over
Child
RevDate: 2025-02-05
CmpDate: 2025-02-05
Utilizing CRISPR-based genetic modification for precise control of seed dormancy: progress, obstacles, and potential directions.
Molecular biology reports, 52(1):204.
Seed dormancy, a complex trait that is influenced by both nuclear and cytoplasmic factors, poses a significant challenge to agricultural productivity. Conventional dormancy-breaking techniques, including mechanical, physiological, and chemical methods, often yield inconsistent results, impair seed quality, and lack precision. This has necessitated exploration of more targeted and efficient approaches. CRISPR-based gene editing has emerged as a promising tool for the precise regulation of seed dormancy without compromising seed viability or sustainability. Although CRISPR has been successfully applied to modify genes that govern physiological traits in various crops, its use in dormancy regulation remains in the early stages. This review examines recent advancements in CRISPR-based approaches for modulating seed dormancy and discusses key gene targets, modification techniques, and the resulting effects. We also consider the future potential of CRISPR to enhance dormancy control across diverse crop species.
Additional Links: PMID-39907946
PubMed:
Citation:
show bibtex listing
hide bibtex listing
@article {pmid39907946,
year = {2025},
author = {Rachappanavar, V},
title = {Utilizing CRISPR-based genetic modification for precise control of seed dormancy: progress, obstacles, and potential directions.},
journal = {Molecular biology reports},
volume = {52},
number = {1},
pages = {204},
pmid = {39907946},
issn = {1573-4978},
mesh = {*Plant Dormancy/genetics ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; *Seeds/genetics ; *Crops, Agricultural/genetics ; Plants, Genetically Modified/genetics ; Gene Expression Regulation, Plant/genetics ; Germination/genetics ; },
abstract = {Seed dormancy, a complex trait that is influenced by both nuclear and cytoplasmic factors, poses a significant challenge to agricultural productivity. Conventional dormancy-breaking techniques, including mechanical, physiological, and chemical methods, often yield inconsistent results, impair seed quality, and lack precision. This has necessitated exploration of more targeted and efficient approaches. CRISPR-based gene editing has emerged as a promising tool for the precise regulation of seed dormancy without compromising seed viability or sustainability. Although CRISPR has been successfully applied to modify genes that govern physiological traits in various crops, its use in dormancy regulation remains in the early stages. This review examines recent advancements in CRISPR-based approaches for modulating seed dormancy and discusses key gene targets, modification techniques, and the resulting effects. We also consider the future potential of CRISPR to enhance dormancy control across diverse crop species.},
}
MeSH Terms:
show MeSH Terms
hide MeSH Terms
*Plant Dormancy/genetics
*Gene Editing/methods
*CRISPR-Cas Systems/genetics
*Seeds/genetics
*Crops, Agricultural/genetics
Plants, Genetically Modified/genetics
Gene Expression Regulation, Plant/genetics
Germination/genetics
RevDate: 2025-02-05
From supplements to superbugs: how probiotic patent gaps drive antimicrobial resistance and the CRISPR-Cas solutions.
Additional Links: PMID-39907302
Publisher:
PubMed:
Citation:
show bibtex listing
hide bibtex listing
@article {pmid39907302,
year = {2025},
author = {Talat, A and Khan, AU},
title = {From supplements to superbugs: how probiotic patent gaps drive antimicrobial resistance and the CRISPR-Cas solutions.},
journal = {Pharmaceutical patent analyst},
volume = {},
number = {},
pages = {1-3},
doi = {10.1080/20468954.2025.2459592},
pmid = {39907302},
issn = {2046-8962},
}
RevDate: 2025-02-05
New frontiers in CRISPR: Addressing antimicrobial resistance with Cas9, Cas12, Cas13, and Cas14.
Heliyon, 11(2):e42013.
BACKGROUND: The issue of antimicrobial resistance (AMR) poses a major challenge to global health, evidenced by alarming mortality predictions and the diminishing efficiency of conventional antimicrobial drugs. The CRISPR-Cas system has proven to be a powerful tool in addressing this challenge. It originated from bacterial adaptive immune mechanisms and has gained significant recognition in the scientific community.
OBJECTIVES: This review aims to explore the applications of CRISPR-Cas technologies in combating AMR, evaluating their effectiveness, challenges, and potential for integration into current antimicrobial strategies.
METHODS: We conducted a comprehensive review of recent literature from databases such as PubMed and Web of Science, focusing on studies that employ CRISPR-Cas technologies against AMR.
CONCLUSIONS: CRISPR-Cas technologies offer a transformative approach to combat AMR, with potential applications that extend beyond traditional antimicrobial strategies. Integrating these technologies with existing methods could significantly enhance our ability to manage and potentially reverse the growing problem of antimicrobial resistance. Future research should address technical and ethical barriers to facilitate safe and effective clinical and environmental applications. This review underscores the necessity for interdisciplinary collaboration and international cooperation to harness the full potential of CRISPR-Cas technologies in the fight against superbugs.
Additional Links: PMID-39906792
PubMed:
Citation:
show bibtex listing
hide bibtex listing
@article {pmid39906792,
year = {2025},
author = {Ali Agha, ASA and Al-Samydai, A and Aburjai, T},
title = {New frontiers in CRISPR: Addressing antimicrobial resistance with Cas9, Cas12, Cas13, and Cas14.},
journal = {Heliyon},
volume = {11},
number = {2},
pages = {e42013},
pmid = {39906792},
issn = {2405-8440},
abstract = {BACKGROUND: The issue of antimicrobial resistance (AMR) poses a major challenge to global health, evidenced by alarming mortality predictions and the diminishing efficiency of conventional antimicrobial drugs. The CRISPR-Cas system has proven to be a powerful tool in addressing this challenge. It originated from bacterial adaptive immune mechanisms and has gained significant recognition in the scientific community.
OBJECTIVES: This review aims to explore the applications of CRISPR-Cas technologies in combating AMR, evaluating their effectiveness, challenges, and potential for integration into current antimicrobial strategies.
METHODS: We conducted a comprehensive review of recent literature from databases such as PubMed and Web of Science, focusing on studies that employ CRISPR-Cas technologies against AMR.
CONCLUSIONS: CRISPR-Cas technologies offer a transformative approach to combat AMR, with potential applications that extend beyond traditional antimicrobial strategies. Integrating these technologies with existing methods could significantly enhance our ability to manage and potentially reverse the growing problem of antimicrobial resistance. Future research should address technical and ethical barriers to facilitate safe and effective clinical and environmental applications. This review underscores the necessity for interdisciplinary collaboration and international cooperation to harness the full potential of CRISPR-Cas technologies in the fight against superbugs.},
}
RevDate: 2025-02-05
Enhancing CRISPR-Cas-based gene targeting in tomato using a dominant-negative ku80.
Horticulture research, 12(2):uhae294.
The CRISPR-Cas-based gene targeting (GT) method has enabled precise modifications of genomic DNA ranging from single base to several kilobase scales through homologous recombination (HR). In plant somatic cells, canonical non-homologous end-joining (cNHEJ) is the predominant mechanism for repairing double-stranded breaks (DSBs), thus limiting the HR-mediated GT. In this study, we implemented an approach to shift the repair pathway preference toward HR by using a dominant-negative ku80 mutant protein (KUDN) to disrupt the initiation of cNHEJ. The employment of KUDN conferred a 1.71- to 3.55-fold improvement in GT efficiency at the callus stage. When we screened transformants, there was a more remarkable increase in GT efficiency, ranging from 1.62- to 9.84-fold, at two specific tomato loci, SlHKT1;2 and SlEPSPS1. With practical levels of efficiency, this enhanced KUDN-based GT tool successfully facilitated a 9-bp addition at an additional locus, SlCAB13. These findings provide another promising method for more efficient and precise plant breeding.
Additional Links: PMID-39906170
PubMed:
Citation:
show bibtex listing
hide bibtex listing
@article {pmid39906170,
year = {2025},
author = {Vu, TV and Nguyen, NT and Kim, J and Vu, MH and Song, YJ and Tran, MT and Sung, YW and Kim, JY},
title = {Enhancing CRISPR-Cas-based gene targeting in tomato using a dominant-negative ku80.},
journal = {Horticulture research},
volume = {12},
number = {2},
pages = {uhae294},
pmid = {39906170},
issn = {2662-6810},
abstract = {The CRISPR-Cas-based gene targeting (GT) method has enabled precise modifications of genomic DNA ranging from single base to several kilobase scales through homologous recombination (HR). In plant somatic cells, canonical non-homologous end-joining (cNHEJ) is the predominant mechanism for repairing double-stranded breaks (DSBs), thus limiting the HR-mediated GT. In this study, we implemented an approach to shift the repair pathway preference toward HR by using a dominant-negative ku80 mutant protein (KUDN) to disrupt the initiation of cNHEJ. The employment of KUDN conferred a 1.71- to 3.55-fold improvement in GT efficiency at the callus stage. When we screened transformants, there was a more remarkable increase in GT efficiency, ranging from 1.62- to 9.84-fold, at two specific tomato loci, SlHKT1;2 and SlEPSPS1. With practical levels of efficiency, this enhanced KUDN-based GT tool successfully facilitated a 9-bp addition at an additional locus, SlCAB13. These findings provide another promising method for more efficient and precise plant breeding.},
}
RevDate: 2025-02-05
Genome editing for grass improvement and future agriculture.
Horticulture research, 12(2):uhae293.
Grasses, including turf and forage, cover most of the earth's surface; predominantly important for land, water, livestock feed, soil, and water conservation, as well as carbon sequestration. Improved production and quality of grasses by modern molecular breeding is gaining more research attention. Recent advances in genome-editing technologies are helping to revolutionize plant breeding and also offering smart and efficient acceleration on grass improvement. Here, we reviewed all recent researches using (CRISPR)/CRISPR-associated protein (Cas)-mediated genome editing tools to enhance the growth and quality of forage and turf grasses. Furthermore, we highlighted emerging approaches aimed at advancing grass breeding program. We assessed the CRISPR-Cas effectiveness, discussed the challenges associated with its application, and explored future perspectives primarily focusing on turf and forage grasses. Despite the promising potential of genome editing in grasses, its current efficiency remains limited due to several bottlenecks, such as the absence of comprehensive reference genomes, the lack of efficient gene delivery tools, unavailability of suitable vector and delivery for grass species, high polyploidization, and multiple homoeoalleles, etc. Despite these challenges, the CRISPR-Cas system holds great potential to fully harness its benefits in grass breeding and genetics, aiming to improve and sustain the quantity and quality of turf and forage grasses.
Additional Links: PMID-39906167
PubMed:
Citation:
show bibtex listing
hide bibtex listing
@article {pmid39906167,
year = {2025},
author = {Bilal, M and Geng, J and Chen, L and García-Caparros, P and Hu, T},
title = {Genome editing for grass improvement and future agriculture.},
journal = {Horticulture research},
volume = {12},
number = {2},
pages = {uhae293},
pmid = {39906167},
issn = {2662-6810},
abstract = {Grasses, including turf and forage, cover most of the earth's surface; predominantly important for land, water, livestock feed, soil, and water conservation, as well as carbon sequestration. Improved production and quality of grasses by modern molecular breeding is gaining more research attention. Recent advances in genome-editing technologies are helping to revolutionize plant breeding and also offering smart and efficient acceleration on grass improvement. Here, we reviewed all recent researches using (CRISPR)/CRISPR-associated protein (Cas)-mediated genome editing tools to enhance the growth and quality of forage and turf grasses. Furthermore, we highlighted emerging approaches aimed at advancing grass breeding program. We assessed the CRISPR-Cas effectiveness, discussed the challenges associated with its application, and explored future perspectives primarily focusing on turf and forage grasses. Despite the promising potential of genome editing in grasses, its current efficiency remains limited due to several bottlenecks, such as the absence of comprehensive reference genomes, the lack of efficient gene delivery tools, unavailability of suitable vector and delivery for grass species, high polyploidization, and multiple homoeoalleles, etc. Despite these challenges, the CRISPR-Cas system holds great potential to fully harness its benefits in grass breeding and genetics, aiming to improve and sustain the quantity and quality of turf and forage grasses.},
}
RevDate: 2025-02-05
CmpDate: 2025-02-05
Increased distribution of carbon metabolic flux during de novo cytidine biosynthesis via attenuation of the acetic acid metabolism pathway in Escherichia coli.
Microbial cell factories, 24(1):36.
Acetic acid, a by-product of cytidine synthesis, competes for carbon flux from central metabolism, which may be directed either to the tricarboxylic acid (TCA) cycle for cytidine synthesis or to overflow metabolites, such as acetic acid. In Escherichia coli, the acetic acid synthesis pathway, regulated by the poxB and pta genes, facilitates carbon consumption during cytidine production. To mitigate carbon source loss, the CRISPR-Cas9 gene-editing technique was employed to knock out the poxB and pta genes in E. coli, generating the engineered strains K12ΔpoxB and K12ΔpoxBΔpta. After 39 h of fermentation in 500 mL shake flasks, the cytidine yields of strains K12ΔpoxB and K12ΔpoxBΔpta were 1.91 ± 0.04 g/L and 18.28 ± 0.22 g/L, respectively. Disruption of the poxB and pta genes resulted in reduced acetic acid production and glucose consumption. Transcriptomic and metabolomic analyses revealed that impairing the acetic acid metabolic pathway in E. coli effectively redirected carbon flux toward cytidine biosynthesis, yielding a 5.26-fold reduction in acetate metabolism and an 11.56-fold increase in cytidine production. These findings provide novel insights into the influence of the acetate metabolic pathway on cytidine biosynthesis in E. coli.
Additional Links: PMID-39905471
PubMed:
Citation:
show bibtex listing
hide bibtex listing
@article {pmid39905471,
year = {2025},
author = {Ye, T and Ding, W and An, Z and Zhang, H and Wei, X and Xu, J and Liu, H and Fang, H},
title = {Increased distribution of carbon metabolic flux during de novo cytidine biosynthesis via attenuation of the acetic acid metabolism pathway in Escherichia coli.},
journal = {Microbial cell factories},
volume = {24},
number = {1},
pages = {36},
pmid = {39905471},
issn = {1475-2859},
support = {31860020//National Natural Science Foundation of China/ ; 022004000010//Ningxia Hui Autonomous Region Youth Top Talent Training Project/ ; No. 2020//The Helanshan Scholars Program of Ningxia University/ ; 2023AAC02030//Natural Science Foundation of Ningxia, China/ ; },
mesh = {*Acetic Acid/metabolism ; *Escherichia coli/metabolism/genetics ; *Carbon/metabolism ; *Cytidine/metabolism ; Metabolic Engineering/methods ; Fermentation ; Escherichia coli Proteins/metabolism/genetics ; Metabolic Networks and Pathways ; Glucose/metabolism ; CRISPR-Cas Systems ; Citric Acid Cycle ; },
abstract = {Acetic acid, a by-product of cytidine synthesis, competes for carbon flux from central metabolism, which may be directed either to the tricarboxylic acid (TCA) cycle for cytidine synthesis or to overflow metabolites, such as acetic acid. In Escherichia coli, the acetic acid synthesis pathway, regulated by the poxB and pta genes, facilitates carbon consumption during cytidine production. To mitigate carbon source loss, the CRISPR-Cas9 gene-editing technique was employed to knock out the poxB and pta genes in E. coli, generating the engineered strains K12ΔpoxB and K12ΔpoxBΔpta. After 39 h of fermentation in 500 mL shake flasks, the cytidine yields of strains K12ΔpoxB and K12ΔpoxBΔpta were 1.91 ± 0.04 g/L and 18.28 ± 0.22 g/L, respectively. Disruption of the poxB and pta genes resulted in reduced acetic acid production and glucose consumption. Transcriptomic and metabolomic analyses revealed that impairing the acetic acid metabolic pathway in E. coli effectively redirected carbon flux toward cytidine biosynthesis, yielding a 5.26-fold reduction in acetate metabolism and an 11.56-fold increase in cytidine production. These findings provide novel insights into the influence of the acetate metabolic pathway on cytidine biosynthesis in E. coli.},
}
MeSH Terms:
show MeSH Terms
hide MeSH Terms
*Acetic Acid/metabolism
*Escherichia coli/metabolism/genetics
*Carbon/metabolism
*Cytidine/metabolism
Metabolic Engineering/methods
Fermentation
Escherichia coli Proteins/metabolism/genetics
Metabolic Networks and Pathways
Glucose/metabolism
CRISPR-Cas Systems
Citric Acid Cycle
RevDate: 2025-02-05
CmpDate: 2025-02-05
Transitioning from wet lab to artificial intelligence: a systematic review of AI predictors in CRISPR.
Journal of translational medicine, 23(1):153.
The revolutionary CRISPR-Cas9 system leverages a programmable guide RNA (gRNA) and Cas9 proteins to precisely cleave problematic regions within DNA sequences. This groundbreaking technology holds immense potential for the development of targeted therapies for a wide range of diseases, including cancers, genetic disorders, and hereditary diseases. CRISPR-Cas9 based genome editing is a multi-step process such as designing a precise gRNA, selecting the appropriate Cas protein, and thoroughly evaluating both on-target and off-target activity of the Cas9-gRNA complex. To ensure the accuracy and effectiveness of CRISPR-Cas9 system, after the targeted DNA cleavage, the process requires careful analysis of the resultant outcomes such as indels and deletions. Following the success of artificial intelligence (AI) in various fields, researchers are now leveraging AI algorithms to catalyze and optimize the multi-step process of CRISPR-Cas9 system. To achieve this goal AI-driven applications are being integrated into each step, but existing AI predictors have limited performance and many steps still rely on expensive and time-consuming wet-lab experiments. The primary reason behind low performance of AI predictors is the gap between CRISPR and AI fields. Effective integration of AI into multi-step CRISPR-Cas9 system demands comprehensive knowledge of both domains. This paper bridges the knowledge gap between AI and CRISPR-Cas9 research. It offers a unique platform for AI researchers to grasp deep understanding of the biological foundations behind each step in the CRISPR-Cas9 multi-step process. Furthermore, it provides details of 80 available CRISPR-Cas9 system-related datasets that can be utilized to develop AI-driven applications. Within the landscape of AI predictors in CRISPR-Cas9 multi-step process, it provides insights of representation learning methods, machine and deep learning methods trends, and performance values of existing 50 predictive pipelines. In the context of representation learning methods and classifiers/regressors, a thorough analysis of existing predictive pipelines is utilized for recommendations to develop more robust and precise predictive pipelines.
Additional Links: PMID-39905452
PubMed:
Citation:
show bibtex listing
hide bibtex listing
@article {pmid39905452,
year = {2025},
author = {Abbasi, AF and Asim, MN and Dengel, A},
title = {Transitioning from wet lab to artificial intelligence: a systematic review of AI predictors in CRISPR.},
journal = {Journal of translational medicine},
volume = {23},
number = {1},
pages = {153},
pmid = {39905452},
issn = {1479-5876},
mesh = {*Artificial Intelligence ; Humans ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; },
abstract = {The revolutionary CRISPR-Cas9 system leverages a programmable guide RNA (gRNA) and Cas9 proteins to precisely cleave problematic regions within DNA sequences. This groundbreaking technology holds immense potential for the development of targeted therapies for a wide range of diseases, including cancers, genetic disorders, and hereditary diseases. CRISPR-Cas9 based genome editing is a multi-step process such as designing a precise gRNA, selecting the appropriate Cas protein, and thoroughly evaluating both on-target and off-target activity of the Cas9-gRNA complex. To ensure the accuracy and effectiveness of CRISPR-Cas9 system, after the targeted DNA cleavage, the process requires careful analysis of the resultant outcomes such as indels and deletions. Following the success of artificial intelligence (AI) in various fields, researchers are now leveraging AI algorithms to catalyze and optimize the multi-step process of CRISPR-Cas9 system. To achieve this goal AI-driven applications are being integrated into each step, but existing AI predictors have limited performance and many steps still rely on expensive and time-consuming wet-lab experiments. The primary reason behind low performance of AI predictors is the gap between CRISPR and AI fields. Effective integration of AI into multi-step CRISPR-Cas9 system demands comprehensive knowledge of both domains. This paper bridges the knowledge gap between AI and CRISPR-Cas9 research. It offers a unique platform for AI researchers to grasp deep understanding of the biological foundations behind each step in the CRISPR-Cas9 multi-step process. Furthermore, it provides details of 80 available CRISPR-Cas9 system-related datasets that can be utilized to develop AI-driven applications. Within the landscape of AI predictors in CRISPR-Cas9 multi-step process, it provides insights of representation learning methods, machine and deep learning methods trends, and performance values of existing 50 predictive pipelines. In the context of representation learning methods and classifiers/regressors, a thorough analysis of existing predictive pipelines is utilized for recommendations to develop more robust and precise predictive pipelines.},
}
MeSH Terms:
show MeSH Terms
hide MeSH Terms
*Artificial Intelligence
Humans
*CRISPR-Cas Systems/genetics
*Gene Editing/methods
Clustered Regularly Interspaced Short Palindromic Repeats/genetics
RevDate: 2025-02-05
CmpDate: 2025-02-05
TAB2 deficiency induces dilated cardiomyopathy by promoting mitochondrial calcium overload in human iPSC-derived cardiomyocytes.
Molecular medicine (Cambridge, Mass.), 31(1):42.
BACKGROUND: TGF-β-activated kinase 1 binding protein 2 (TAB2) is an intermediary protein that links Tumor necrosis factor receptor 1 (TNFR1) and other receptor signals to the TGF-β-activated kinase 1 (TAK1) signaling complex. TAB2 frameshift mutations have been linked to dilated cardiomyopathy (DCM), while the exact mechanism needs further investigation.
METHODS: In this study, we generated a TAB2 compound heterozygous knockout cell line in induced pluripotent stem cells (iPSCs) derived from a healthy individual using CRISPR/Cas9 technology. IPSCs are not species-dependent, are readily accessible, and raise fewer ethical concerns.
RESULTS: TAB2 disruption had no impact on the cardiac differentiation of iPSCs and led to confirmed TAB2 deficiency in human iPSC-derived cardiomyocytes (hiPSC-CMs). TAB2-deficient hiPSC-CMs were found to develop phenotypic features of DCM, such as distorted sarcomeric ultrastructure, decreased contractility and energy production, and mitochondrial damage at day 30 post differentiation. Paradoxically, TAB2 knockout cell lines showed abnormal calcium handling after 40 days, later than reduced contractility, suggesting that the main cause of impaired contractility was abnormal energy production due to mitochondrial damage. As early as day 25, TAB2 knockout cardiomyocytes showed significant mitochondrial calcium overload, which can lead to mitochondrial damage. Furthermore, TAB2 knockout activated receptor-interacting protein kinase 1 (RIPK1), leading to an increase in mitochondrial calcium uniporter (MCU) expression, thereby augmenting the uptake of mitochondrial calcium ions. Finally, the application of the RIPK1 inhibitor Nec-1s prevents the progression of these phenotypes.
CONCLUSIONS: In summary, TAB2 abatement cardiomyocytes mimic dilated cardiomyopathy in vitro. This finding emphasizes the importance of using a human model to study the underlying mechanisms of this specific disease. More importantly, the discovery of a unique pathogenic pathway introduces a new notion for the future management of dilated cardiomyopathy.
Additional Links: PMID-39905300
PubMed:
Citation:
show bibtex listing
hide bibtex listing
@article {pmid39905300,
year = {2025},
author = {Sun, W and Zhang, J and Li, S and Fu, W and Liu, Y and Liu, M and Dong, J and Zhao, X and Li, X},
title = {TAB2 deficiency induces dilated cardiomyopathy by promoting mitochondrial calcium overload in human iPSC-derived cardiomyocytes.},
journal = {Molecular medicine (Cambridge, Mass.)},
volume = {31},
number = {1},
pages = {42},
pmid = {39905300},
issn = {1528-3658},
support = {82000352//National Natural Science Foundation of China/ ; 82000352//National Natural Science Foundation of China/ ; 82000352//National Natural Science Foundation of China/ ; 82000352//National Natural Science Foundation of China/ ; 82000352//National Natural Science Foundation of China/ ; 82000352//National Natural Science Foundation of China/ ; 82000352//National Natural Science Foundation of China/ ; 82000352//National Natural Science Foundation of China/ ; 82000352//National Natural Science Foundation of China/ ; 2020M672295//China Postdoctoral Science Foundation/ ; 2020M672295//China Postdoctoral Science Foundation/ ; 2020M672295//China Postdoctoral Science Foundation/ ; 2020M672295//China Postdoctoral Science Foundation/ ; 2020M672295//China Postdoctoral Science Foundation/ ; 2020M672295//China Postdoctoral Science Foundation/ ; 2020M672295//China Postdoctoral Science Foundation/ ; 2020M672295//China Postdoctoral Science Foundation/ ; 2020M672295//China Postdoctoral Science Foundation/ ; },
mesh = {Humans ; *Myocytes, Cardiac/metabolism ; *Cardiomyopathy, Dilated/metabolism/genetics/pathology ; *Induced Pluripotent Stem Cells/metabolism ; *Adaptor Proteins, Signal Transducing/genetics/metabolism ; *Calcium/metabolism ; Cell Differentiation ; Mitochondria/metabolism ; Gene Knockout Techniques ; Cell Line ; CRISPR-Cas Systems ; },
abstract = {BACKGROUND: TGF-β-activated kinase 1 binding protein 2 (TAB2) is an intermediary protein that links Tumor necrosis factor receptor 1 (TNFR1) and other receptor signals to the TGF-β-activated kinase 1 (TAK1) signaling complex. TAB2 frameshift mutations have been linked to dilated cardiomyopathy (DCM), while the exact mechanism needs further investigation.
METHODS: In this study, we generated a TAB2 compound heterozygous knockout cell line in induced pluripotent stem cells (iPSCs) derived from a healthy individual using CRISPR/Cas9 technology. IPSCs are not species-dependent, are readily accessible, and raise fewer ethical concerns.
RESULTS: TAB2 disruption had no impact on the cardiac differentiation of iPSCs and led to confirmed TAB2 deficiency in human iPSC-derived cardiomyocytes (hiPSC-CMs). TAB2-deficient hiPSC-CMs were found to develop phenotypic features of DCM, such as distorted sarcomeric ultrastructure, decreased contractility and energy production, and mitochondrial damage at day 30 post differentiation. Paradoxically, TAB2 knockout cell lines showed abnormal calcium handling after 40 days, later than reduced contractility, suggesting that the main cause of impaired contractility was abnormal energy production due to mitochondrial damage. As early as day 25, TAB2 knockout cardiomyocytes showed significant mitochondrial calcium overload, which can lead to mitochondrial damage. Furthermore, TAB2 knockout activated receptor-interacting protein kinase 1 (RIPK1), leading to an increase in mitochondrial calcium uniporter (MCU) expression, thereby augmenting the uptake of mitochondrial calcium ions. Finally, the application of the RIPK1 inhibitor Nec-1s prevents the progression of these phenotypes.
CONCLUSIONS: In summary, TAB2 abatement cardiomyocytes mimic dilated cardiomyopathy in vitro. This finding emphasizes the importance of using a human model to study the underlying mechanisms of this specific disease. More importantly, the discovery of a unique pathogenic pathway introduces a new notion for the future management of dilated cardiomyopathy.},
}
MeSH Terms:
show MeSH Terms
hide MeSH Terms
Humans
*Myocytes, Cardiac/metabolism
*Cardiomyopathy, Dilated/metabolism/genetics/pathology
*Induced Pluripotent Stem Cells/metabolism
*Adaptor Proteins, Signal Transducing/genetics/metabolism
*Calcium/metabolism
Cell Differentiation
Mitochondria/metabolism
Gene Knockout Techniques
Cell Line
CRISPR-Cas Systems
RevDate: 2025-02-05
CmpDate: 2025-02-05
Multi-locus CRISPRi targeting with a single truncated guide RNA.
Nature communications, 16(1):1357.
A critical goal in functional genomics is evaluating which non-coding elements contribute to gene expression, cellular function, and disease. Functional characterization remains a challenge due to the abundance and complexity of candidate elements. Here, we develop a CRISPRi-based approach for multi-locus screening of putative transcription factor binding sites with a single truncated guide. A truncated guide with hundreds of sequence match sites can reliably disrupt enhancer activity, which expands the targeting scope of CRISPRi while maintaining repressive efficacy. We screen over 13,000 possible CTCF binding sites with 24 guides at 10 nucleotides in spacer length. These truncated guides direct CRISPRi-mediated deposition of repressive H3K9me3 marks and disrupt transcription factor binding at most sequence match target sites. This approach can be a valuable screening step for testing transcription factor binding motifs or other repeated genomic sequences and is easily implemented with existing tools.
Additional Links: PMID-39905017
PubMed:
Citation:
show bibtex listing
hide bibtex listing
@article {pmid39905017,
year = {2025},
author = {Moore, MM and Wekhande, S and Issner, R and Collins, A and Cruz, AJ and Liu, YV and Javed, N and Casaní-Galdón, S and Buenrostro, JD and Epstein, CB and Mattei, E and Doench, JG and Bernstein, BE and Shoresh, N and Najm, FJ},
title = {Multi-locus CRISPRi targeting with a single truncated guide RNA.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {1357},
pmid = {39905017},
issn = {2041-1723},
mesh = {*RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; Humans ; Binding Sites/genetics ; *CRISPR-Cas Systems ; Transcription Factors/metabolism/genetics ; CCCTC-Binding Factor/metabolism/genetics ; Enhancer Elements, Genetic/genetics ; Genetic Loci ; Histones/metabolism/genetics ; },
abstract = {A critical goal in functional genomics is evaluating which non-coding elements contribute to gene expression, cellular function, and disease. Functional characterization remains a challenge due to the abundance and complexity of candidate elements. Here, we develop a CRISPRi-based approach for multi-locus screening of putative transcription factor binding sites with a single truncated guide. A truncated guide with hundreds of sequence match sites can reliably disrupt enhancer activity, which expands the targeting scope of CRISPRi while maintaining repressive efficacy. We screen over 13,000 possible CTCF binding sites with 24 guides at 10 nucleotides in spacer length. These truncated guides direct CRISPRi-mediated deposition of repressive H3K9me3 marks and disrupt transcription factor binding at most sequence match target sites. This approach can be a valuable screening step for testing transcription factor binding motifs or other repeated genomic sequences and is easily implemented with existing tools.},
}
MeSH Terms:
show MeSH Terms
hide MeSH Terms
*RNA, Guide, CRISPR-Cas Systems/genetics/metabolism
Humans
Binding Sites/genetics
*CRISPR-Cas Systems
Transcription Factors/metabolism/genetics
CCCTC-Binding Factor/metabolism/genetics
Enhancer Elements, Genetic/genetics
Genetic Loci
Histones/metabolism/genetics
RevDate: 2025-02-05
CmpDate: 2025-02-05
Structural basis for RNA-guided DNA degradation by Cas5-HNH/Cascade complex.
Nature communications, 16(1):1335.
Type I-E CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated proteins) system is one of the most extensively studied RNA-guided adaptive immune systems in prokaryotes, providing defense against foreign genetic elements. Unlike the previously characterized Cas3 nuclease, which exhibits progressive DNA cleavage in the typical type I-E system, a recently identified HNH-comprising Cascade system enables precise DNA cleavage. Here, we present several near-atomic cryo-electron microscopy (cryo-EM) structures of the Candidatus Cloacimonetes bacterium Cas5-HNH/Cascade complex, both in its DNA-bound and unbound states. Our analysis reveals extensive interactions between the HNH domain and adjacent subunits, including Cas6 and Cas11, with mutations in these key interactions significantly impairing enzymatic activity. Upon DNA binding, the Cas5-HNH/Cascade complex adopts a more compact conformation, with subunits converging toward the center of nuclease, leading to its activation. Notably, we also find that divalent ions such as zinc, cobalt, and nickel down-regulate enzyme activity by destabilizing the Cascade complex. Together, these findings offer structural insights into the assembly and activation of the Cas5-HNH/Cascade complex.
Additional Links: PMID-39904990
PubMed:
Citation:
show bibtex listing
hide bibtex listing
@article {pmid39904990,
year = {2025},
author = {Liu, Y and Wang, L and Zhang, Q and Fu, P and Zhang, L and Yu, Y and Zhang, H and Zhu, H},
title = {Structural basis for RNA-guided DNA degradation by Cas5-HNH/Cascade complex.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {1335},
pmid = {39904990},
issn = {2041-1723},
mesh = {*Cryoelectron Microscopy ; *CRISPR-Associated Proteins/metabolism/chemistry ; *CRISPR-Cas Systems ; *DNA/metabolism/chemistry ; RNA, Guide, CRISPR-Cas Systems/metabolism ; Bacterial Proteins/metabolism/chemistry/genetics ; Models, Molecular ; Mutation ; Protein Binding ; DNA Cleavage ; Protein Domains ; },
abstract = {Type I-E CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated proteins) system is one of the most extensively studied RNA-guided adaptive immune systems in prokaryotes, providing defense against foreign genetic elements. Unlike the previously characterized Cas3 nuclease, which exhibits progressive DNA cleavage in the typical type I-E system, a recently identified HNH-comprising Cascade system enables precise DNA cleavage. Here, we present several near-atomic cryo-electron microscopy (cryo-EM) structures of the Candidatus Cloacimonetes bacterium Cas5-HNH/Cascade complex, both in its DNA-bound and unbound states. Our analysis reveals extensive interactions between the HNH domain and adjacent subunits, including Cas6 and Cas11, with mutations in these key interactions significantly impairing enzymatic activity. Upon DNA binding, the Cas5-HNH/Cascade complex adopts a more compact conformation, with subunits converging toward the center of nuclease, leading to its activation. Notably, we also find that divalent ions such as zinc, cobalt, and nickel down-regulate enzyme activity by destabilizing the Cascade complex. Together, these findings offer structural insights into the assembly and activation of the Cas5-HNH/Cascade complex.},
}
MeSH Terms:
show MeSH Terms
hide MeSH Terms
*Cryoelectron Microscopy
*CRISPR-Associated Proteins/metabolism/chemistry
*CRISPR-Cas Systems
*DNA/metabolism/chemistry
RNA, Guide, CRISPR-Cas Systems/metabolism
Bacterial Proteins/metabolism/chemistry/genetics
Models, Molecular
Mutation
Protein Binding
DNA Cleavage
Protein Domains
RevDate: 2025-02-04
An electrochemiluminescence strategy with proximity ligation triggered multiple catalytic hairpin assembly induced CRISPR/Cas 12a system for analysis of paraquat.
Bioelectrochemistry (Amsterdam, Netherlands), 164:108915 pii:S1567-5394(25)00018-0 [Epub ahead of print].
Paraquat (PQ) as a widely used non-selective herbicides has gained attention in agricultural residue detection and food safety. Herein, a novel quantitative analysis approach for PQ was proposed based on a novel kind of aggregation-induced emission electrochemiluminescence (AIECL) emitters, tetraphenylethylene-luminol (TPE-L) with a small molecule-induced multiple catalytic hairpin assembly (CHA) amplification strategy, the competitive immune reaction and CRISPR/Cas12a system. The target molecule PQ is introduced into a signal cycle, and auxiliary sensitization cycles are constructed by virtue of the cleavage characteristics of the CRISPR/Cas12a system, which realized the multiple utilization of the target by using both cis- and trans-cleavage activities. In addition, the new multiple CHA amplification strategy was attributed to cross-catalytic hairpin assembly caused by the products of the CHA cycle as the initiator chain of the next CHA cycle, realizing the efficient utilization of cyclic products and producing high-efficiency signal amplification. Thus, the ECL biosensor for ultrasensitive analysis of PQ was successfully constructed with a limit of detection of 0.7 pg/mL. Importantly, it could be easily-extended to other small molecules simply by replacing paired antibodies, providing prospects in agricultural residue detection, food safety and related medical applications.
Additional Links: PMID-39904298
Publisher:
PubMed:
Citation:
show bibtex listing
hide bibtex listing
@article {pmid39904298,
year = {2025},
author = {Zhong, WJ and Yang, WG and Zhang, Y and Li, T and Su, ML and Yuan, R and Xu, S and Liang, WB},
title = {An electrochemiluminescence strategy with proximity ligation triggered multiple catalytic hairpin assembly induced CRISPR/Cas 12a system for analysis of paraquat.},
journal = {Bioelectrochemistry (Amsterdam, Netherlands)},
volume = {164},
number = {},
pages = {108915},
doi = {10.1016/j.bioelechem.2025.108915},
pmid = {39904298},
issn = {1878-562X},
abstract = {Paraquat (PQ) as a widely used non-selective herbicides has gained attention in agricultural residue detection and food safety. Herein, a novel quantitative analysis approach for PQ was proposed based on a novel kind of aggregation-induced emission electrochemiluminescence (AIECL) emitters, tetraphenylethylene-luminol (TPE-L) with a small molecule-induced multiple catalytic hairpin assembly (CHA) amplification strategy, the competitive immune reaction and CRISPR/Cas12a system. The target molecule PQ is introduced into a signal cycle, and auxiliary sensitization cycles are constructed by virtue of the cleavage characteristics of the CRISPR/Cas12a system, which realized the multiple utilization of the target by using both cis- and trans-cleavage activities. In addition, the new multiple CHA amplification strategy was attributed to cross-catalytic hairpin assembly caused by the products of the CHA cycle as the initiator chain of the next CHA cycle, realizing the efficient utilization of cyclic products and producing high-efficiency signal amplification. Thus, the ECL biosensor for ultrasensitive analysis of PQ was successfully constructed with a limit of detection of 0.7 pg/mL. Importantly, it could be easily-extended to other small molecules simply by replacing paired antibodies, providing prospects in agricultural residue detection, food safety and related medical applications.},
}
RevDate: 2025-02-04
CmpDate: 2025-02-04
Targeting pseudoknots with Cas13b inhibits porcine epidemic diarrhoea virus replication.
The Journal of general virology, 106(2):.
Clustered regularly interspaced short palindromic repeats-associated protein 13 (CRISPR-Cas13), an RNA editing technology, has shown potential in combating RNA viruses by degrading viral RNA within mammalian cells. In this study, we demonstrate the effective inhibition of porcine epidemic diarrhoea virus (PEDV) replication and spread using CRISPR-Cas13. We analysed the sequence similarity of the pseudoknot region between PEDV and severe acute respiratory syndrome coronavirus 2, both belonging to the Coronaviridae family, as well as the similarity of the RNA-dependent RNA polymerase (RdRp) gene region among three different strains of the PED virus. Based on this analysis, we synthesized three CRISPR RNAs (crRNAs) targeting the pseudoknot region and the nonpseudoknot region, each for comparison. In cells treated with crRNA #3 targeting the pseudoknot region, RdRp gene expression decreased by 95%, membrane (M) gene expression by 89% and infectious PEDV titre within the cells reduced by over 95%. Additionally, PED viral nucleocapsid (N) and M protein expression levels decreased by 83 and 98%, respectively. The optimal concentration for high antiviral efficacy without cytotoxicity was determined. Treating cells with 1.5 µg of Cas13b mRNA and 0.5 µg of crRNA resulted in no cytotoxicity while achieving over 95% inhibition of PEDV replication. The Cas13b mRNA therapeutics approach was validated as significantly more effective through a comparative study with merafloxacin, a drug targeting the pseudoknot region of the viral genome. Our results indicate that the pseudoknot region plays a crucial role in the degradation of the PEDV genome through the CRISPR-Cas13 system. Therefore, targeting Cas13b to the pseudoknot offers a promising new approach for treating coronavirus infections.
Additional Links: PMID-39903512
PubMed:
Citation:
show bibtex listing
hide bibtex listing
@article {pmid39903512,
year = {2025},
author = {Han, HJ and Yu, D and Yu, J and Kim, J and Do Heo, W and Tark, D and Kang, SM},
title = {Targeting pseudoknots with Cas13b inhibits porcine epidemic diarrhoea virus replication.},
journal = {The Journal of general virology},
volume = {106},
number = {2},
pages = {},
pmid = {39903512},
issn = {1465-2099},
mesh = {*Porcine epidemic diarrhea virus/genetics/physiology ; *Virus Replication/drug effects ; Animals ; Chlorocebus aethiops ; *CRISPR-Cas Systems ; Swine ; Vero Cells ; RNA, Viral/genetics/metabolism ; CRISPR-Associated Proteins/genetics/metabolism ; Swine Diseases/virology ; RNA-Dependent RNA Polymerase/genetics/metabolism ; Antiviral Agents/pharmacology ; },
abstract = {Clustered regularly interspaced short palindromic repeats-associated protein 13 (CRISPR-Cas13), an RNA editing technology, has shown potential in combating RNA viruses by degrading viral RNA within mammalian cells. In this study, we demonstrate the effective inhibition of porcine epidemic diarrhoea virus (PEDV) replication and spread using CRISPR-Cas13. We analysed the sequence similarity of the pseudoknot region between PEDV and severe acute respiratory syndrome coronavirus 2, both belonging to the Coronaviridae family, as well as the similarity of the RNA-dependent RNA polymerase (RdRp) gene region among three different strains of the PED virus. Based on this analysis, we synthesized three CRISPR RNAs (crRNAs) targeting the pseudoknot region and the nonpseudoknot region, each for comparison. In cells treated with crRNA #3 targeting the pseudoknot region, RdRp gene expression decreased by 95%, membrane (M) gene expression by 89% and infectious PEDV titre within the cells reduced by over 95%. Additionally, PED viral nucleocapsid (N) and M protein expression levels decreased by 83 and 98%, respectively. The optimal concentration for high antiviral efficacy without cytotoxicity was determined. Treating cells with 1.5 µg of Cas13b mRNA and 0.5 µg of crRNA resulted in no cytotoxicity while achieving over 95% inhibition of PEDV replication. The Cas13b mRNA therapeutics approach was validated as significantly more effective through a comparative study with merafloxacin, a drug targeting the pseudoknot region of the viral genome. Our results indicate that the pseudoknot region plays a crucial role in the degradation of the PEDV genome through the CRISPR-Cas13 system. Therefore, targeting Cas13b to the pseudoknot offers a promising new approach for treating coronavirus infections.},
}
MeSH Terms:
show MeSH Terms
hide MeSH Terms
*Porcine epidemic diarrhea virus/genetics/physiology
*Virus Replication/drug effects
Animals
Chlorocebus aethiops
*CRISPR-Cas Systems
Swine
Vero Cells
RNA, Viral/genetics/metabolism
CRISPR-Associated Proteins/genetics/metabolism
Swine Diseases/virology
RNA-Dependent RNA Polymerase/genetics/metabolism
Antiviral Agents/pharmacology
RevDate: 2025-02-05
CmpDate: 2025-02-05
Enhanced production of recombinant calf chymosin in Kluyveromyces lactis via CRISPR-Cas9 engineering.
Bioresource technology, 419:132116.
As an important industrial enzyme, chymosin has been widely used in cheese manufacturing. Fermentation with Kluyveromyces lactis has allowed recombinant chymosin production to fit the growing global demand for cheese consumption; yet improvements can be made to allow for stable and larger-scale production. In this work, various chymosin producing (CP) strains were constructed via targeted chromosomal integration of various copies of a prochymosin expression cassette (PEC) using a CRISPR-Cas9 platform optimized for K. lactis. It enabled the demonstration that chymosin yields could be increased along with gradual chromosomal accumulation of PEC inserts within up to 3 copies. Finally, an optimal CP3i strain was constructed, and with which high yields of recombinant chymosin were attained, reaching ca. 1,200 SU/mL in shake-flask fermentation and ca. 28,000 SU/mL in batch-mode bioreaction, respectively. The activity of the product in milk-curding was observed. These findings provide direction to apply K. lactis-based platforms in the subsequent industrial-scale production of recombinant chymosin.
Additional Links: PMID-39863179
Publisher:
PubMed:
Citation:
show bibtex listing
hide bibtex listing
@article {pmid39863179,
year = {2025},
author = {Zheng, Y and Wang, S and Deng, Y and Hu, P and Xue, Q and Li, J and Lei, L and Chan, Z and Yang, J and Peng, W},
title = {Enhanced production of recombinant calf chymosin in Kluyveromyces lactis via CRISPR-Cas9 engineering.},
journal = {Bioresource technology},
volume = {419},
number = {},
pages = {132116},
doi = {10.1016/j.biortech.2025.132116},
pmid = {39863179},
issn = {1873-2976},
mesh = {*Kluyveromyces/genetics/metabolism ; *Chymosin/metabolism/genetics ; *CRISPR-Cas Systems ; *Recombinant Proteins/genetics/metabolism ; Animals ; Cattle ; Fermentation ; },
abstract = {As an important industrial enzyme, chymosin has been widely used in cheese manufacturing. Fermentation with Kluyveromyces lactis has allowed recombinant chymosin production to fit the growing global demand for cheese consumption; yet improvements can be made to allow for stable and larger-scale production. In this work, various chymosin producing (CP) strains were constructed via targeted chromosomal integration of various copies of a prochymosin expression cassette (PEC) using a CRISPR-Cas9 platform optimized for K. lactis. It enabled the demonstration that chymosin yields could be increased along with gradual chromosomal accumulation of PEC inserts within up to 3 copies. Finally, an optimal CP3i strain was constructed, and with which high yields of recombinant chymosin were attained, reaching ca. 1,200 SU/mL in shake-flask fermentation and ca. 28,000 SU/mL in batch-mode bioreaction, respectively. The activity of the product in milk-curding was observed. These findings provide direction to apply K. lactis-based platforms in the subsequent industrial-scale production of recombinant chymosin.},
}
MeSH Terms:
show MeSH Terms
hide MeSH Terms
*Kluyveromyces/genetics/metabolism
*Chymosin/metabolism/genetics
*CRISPR-Cas Systems
*Recombinant Proteins/genetics/metabolism
Animals
Cattle
Fermentation
RevDate: 2025-02-05
CmpDate: 2025-02-05
ParSite is a multicolor DNA labeling system that allows for simultaneous imaging of triple genomic loci in living cells.
PLoS biology, 23(1):e3003009 pii:PBIOLOGY-D-24-01651.
The organization of the human genome in space and time is critical for transcriptional regulation and cell fate determination. However, robust methods for tracking genome organization or genomic interactions over time in living cells are lacking. Here, we developed a multicolor DNA labeling system, ParSite, to simultaneously track triple genomic loci in the U2OS cells. The tricolor ParSite system is derived from the T. thermophilus ParB/ParSc (TtParB/ParSc) system by rational design. We mutated the interface between TtParB and ParSc and generated a new pair of TtParBm and ParSm for genomic DNA labeling. The insertions of 16 base-pair palindromic ParSc and ParSm into genomic loci allow dual-color DNA imaging in living cells. A pair of genomic loci labeled by ParSite could be colocalized with p53-binding protein 1 (53BP1) in response to CRISPR/Cas9-mediated double-strand breaks (DSBs). The ParSite permits tracking promoter and terminator dynamics of the APP gene, which spans 290 kilobases in length. Intriguingly, the hybrid ParS (ParSh) of half-ParSc and half-ParSm enables for the visualization of a third locus independent of ParSc or ParSm. We simultaneously labeled 3 loci with a genomic distance of 36, 89, and 352 kilobases downstream the C3 repeat locus, respectively. In sum, the ParSite is a robust DNA labeling system for tracking multiple genomic loci in space and time in living cells.
Additional Links: PMID-39854604
Publisher:
PubMed:
Citation:
show bibtex listing
hide bibtex listing
@article {pmid39854604,
year = {2025},
author = {He, X and Sun, Y and Ma, H},
title = {ParSite is a multicolor DNA labeling system that allows for simultaneous imaging of triple genomic loci in living cells.},
journal = {PLoS biology},
volume = {23},
number = {1},
pages = {e3003009},
doi = {10.1371/journal.pbio.3003009},
pmid = {39854604},
issn = {1545-7885},
mesh = {Humans ; *CRISPR-Cas Systems ; Genome, Human ; DNA/genetics/metabolism ; Genetic Loci ; Cell Line, Tumor ; Tumor Suppressor p53-Binding Protein 1/metabolism/genetics ; DNA Breaks, Double-Stranded ; Staining and Labeling/methods ; },
abstract = {The organization of the human genome in space and time is critical for transcriptional regulation and cell fate determination. However, robust methods for tracking genome organization or genomic interactions over time in living cells are lacking. Here, we developed a multicolor DNA labeling system, ParSite, to simultaneously track triple genomic loci in the U2OS cells. The tricolor ParSite system is derived from the T. thermophilus ParB/ParSc (TtParB/ParSc) system by rational design. We mutated the interface between TtParB and ParSc and generated a new pair of TtParBm and ParSm for genomic DNA labeling. The insertions of 16 base-pair palindromic ParSc and ParSm into genomic loci allow dual-color DNA imaging in living cells. A pair of genomic loci labeled by ParSite could be colocalized with p53-binding protein 1 (53BP1) in response to CRISPR/Cas9-mediated double-strand breaks (DSBs). The ParSite permits tracking promoter and terminator dynamics of the APP gene, which spans 290 kilobases in length. Intriguingly, the hybrid ParS (ParSh) of half-ParSc and half-ParSm enables for the visualization of a third locus independent of ParSc or ParSm. We simultaneously labeled 3 loci with a genomic distance of 36, 89, and 352 kilobases downstream the C3 repeat locus, respectively. In sum, the ParSite is a robust DNA labeling system for tracking multiple genomic loci in space and time in living cells.},
}
MeSH Terms:
show MeSH Terms
hide MeSH Terms
Humans
*CRISPR-Cas Systems
Genome, Human
DNA/genetics/metabolism
Genetic Loci
Cell Line, Tumor
Tumor Suppressor p53-Binding Protein 1/metabolism/genetics
DNA Breaks, Double-Stranded
Staining and Labeling/methods
RevDate: 2025-02-05
CmpDate: 2025-02-05
Long-term correction of hemophilia A via integration of a functionally enhanced FVIII gene into the AAVS1 locus by nickase in patient-derived iPSCs.
Experimental & molecular medicine, 57(1):184-192.
Hemophilia A (HA) is caused by mutations in coagulation factor VIII (FVIII). Genome editing in conjunction with patient-derived induced pluripotent stem cells (iPSCs) is a promising cell therapy strategy, as it replaces dysfunctional proteins resulting from genetic mutations with normal proteins. However, the low expression level and short half-life of FVIII still remain significant limiting factors in the efficacy of these approaches in HA. Here, we constructed a functionally enhanced FVIII variant, F309S/E1984V-mutated B domain-deleted (BDD)-FVIII (FE-FVIII), with increased activity and stability. We inserted FE-FVIII with a human elongation factor-1 alpha (EF1α) promoter into the AAVS1 locus of HA patient-derived iPSCs via CRISPR/Cas9 (D10A) nickase to ensure expression in any cell type. FE-FVIII was expressed not only in undifferentiated FE-FVIII-inserted (FE-KI) iPSCs but also in endothelial cells (ECs) differentiated from them in vitro. Compared with mice transplanted with wild-type BDD-FVIII-containing ECs, immunocompetent HA mice intravenously transplanted with FE-KI ECs presented a 2.12-fold increase in FVIII activity in the blood and an approximately 20% greater survival rate after hemorrhagic tail injury. For sustained efficacy, FE-KI ECs were subcutaneously transplanted into immunodeficient HA mice, resulting in amelioration of the hemophilia phenotype for more than 3 months. This strategy can improve FVIII function and may provide a universal therapeutic approach for treating HA.
Additional Links: PMID-39762408
PubMed:
Citation:
show bibtex listing
hide bibtex listing
@article {pmid39762408,
year = {2025},
author = {Kim, DH and Choi, SH and Sung, JJ and Kim, S and Yi, H and Park, S and Park, CW and Oh, YW and Lee, J and Kim, DS and Kim, JH and Park, CY and Kim, DW},
title = {Long-term correction of hemophilia A via integration of a functionally enhanced FVIII gene into the AAVS1 locus by nickase in patient-derived iPSCs.},
journal = {Experimental & molecular medicine},
volume = {57},
number = {1},
pages = {184-192},
pmid = {39762408},
issn = {2092-6413},
support = {2022R1A2C2091165//National Research Foundation of Korea (NRF)/ ; 2022R1A2C1091800//National Research Foundation of Korea (NRF)/ ; },
mesh = {*Induced Pluripotent Stem Cells/metabolism/cytology ; *Hemophilia A/therapy/genetics ; *Factor VIII/genetics/metabolism ; Humans ; Animals ; Mice ; *Genetic Therapy/methods ; Gene Editing/methods ; Peptide Elongation Factor 1/genetics/metabolism ; Disease Models, Animal ; Endothelial Cells/metabolism ; Genetic Vectors/genetics ; Dependovirus/genetics ; Genetic Loci ; Cell Differentiation ; CRISPR-Cas Systems ; Mutation ; },
abstract = {Hemophilia A (HA) is caused by mutations in coagulation factor VIII (FVIII). Genome editing in conjunction with patient-derived induced pluripotent stem cells (iPSCs) is a promising cell therapy strategy, as it replaces dysfunctional proteins resulting from genetic mutations with normal proteins. However, the low expression level and short half-life of FVIII still remain significant limiting factors in the efficacy of these approaches in HA. Here, we constructed a functionally enhanced FVIII variant, F309S/E1984V-mutated B domain-deleted (BDD)-FVIII (FE-FVIII), with increased activity and stability. We inserted FE-FVIII with a human elongation factor-1 alpha (EF1α) promoter into the AAVS1 locus of HA patient-derived iPSCs via CRISPR/Cas9 (D10A) nickase to ensure expression in any cell type. FE-FVIII was expressed not only in undifferentiated FE-FVIII-inserted (FE-KI) iPSCs but also in endothelial cells (ECs) differentiated from them in vitro. Compared with mice transplanted with wild-type BDD-FVIII-containing ECs, immunocompetent HA mice intravenously transplanted with FE-KI ECs presented a 2.12-fold increase in FVIII activity in the blood and an approximately 20% greater survival rate after hemorrhagic tail injury. For sustained efficacy, FE-KI ECs were subcutaneously transplanted into immunodeficient HA mice, resulting in amelioration of the hemophilia phenotype for more than 3 months. This strategy can improve FVIII function and may provide a universal therapeutic approach for treating HA.},
}
MeSH Terms:
show MeSH Terms
hide MeSH Terms
*Induced Pluripotent Stem Cells/metabolism/cytology
*Hemophilia A/therapy/genetics
*Factor VIII/genetics/metabolism
Humans
Animals
Mice
*Genetic Therapy/methods
Gene Editing/methods
Peptide Elongation Factor 1/genetics/metabolism
Disease Models, Animal
Endothelial Cells/metabolism
Genetic Vectors/genetics
Dependovirus/genetics
Genetic Loci
Cell Differentiation
CRISPR-Cas Systems
Mutation
RevDate: 2025-02-05
CmpDate: 2025-02-05
Guanidyl-rich α-helical polypeptide enables efficient cytosolic pro-protein delivery and CRISPR-Cas9 genome editing.
Journal of materials chemistry. B, 13(6):1991-2002.
Intracellular delivery of proteins has attracted significant interest in biological research and cancer treatment, yet it continues to face challenges due to the lack of effective delivery approaches. Herein, we developed an efficient strategy via cationic α-helical polypeptide-mediated anionic proprotein delivery. The protein was reversibly modified with adenosine triphosphate via dynamic covalent chemistry to prepare an anionic proprotein (A-protein) with abundant phosphate groups. A guanidyl-decorated α-helical polypeptide (LPP) was employed not only to encapsulate A-protein through electrostatic attraction and hydrogen bonding, forming stable nanocomplexes, but also to enhance cell membrane penetration due to its rigid α-helical conformation. Consequently, this strategy mediated the effective delivery of various proteins with different isoelectric points and molecular weights, including α-chymotrypsin, bovine serum albumin, ribonuclease A, cytochrome C, saporin, horseradish peroxidase, β-galactosidase, and anti-phospho-Akt, into cancer cells. More importantly, it enabled efficient delivery of CRISPR-Cas9 ribonucleoproteins to elicit robust polo-like kinase 1 genome editing for inhibiting cancer cell growth. This rationally designed protein delivery system may benefit the development of intracellular protein-based cancer therapy.
Additional Links: PMID-39760520
Publisher:
PubMed:
Citation:
show bibtex listing
hide bibtex listing
@article {pmid39760520,
year = {2025},
author = {Zhao, Z and Zhang, H and Li, W and Wang, Y and Wang, Y and Yang, H and Yin, L and Liu, X},
title = {Guanidyl-rich α-helical polypeptide enables efficient cytosolic pro-protein delivery and CRISPR-Cas9 genome editing.},
journal = {Journal of materials chemistry. B},
volume = {13},
number = {6},
pages = {1991-2002},
doi = {10.1039/d4tb02009j},
pmid = {39760520},
issn = {2050-7518},
mesh = {Humans ; *Gene Editing ; *CRISPR-Cas Systems/genetics ; *Peptides/chemistry/pharmacology ; Guanidine/chemistry ; Cytosol/metabolism ; Protein Conformation, alpha-Helical ; },
abstract = {Intracellular delivery of proteins has attracted significant interest in biological research and cancer treatment, yet it continues to face challenges due to the lack of effective delivery approaches. Herein, we developed an efficient strategy via cationic α-helical polypeptide-mediated anionic proprotein delivery. The protein was reversibly modified with adenosine triphosphate via dynamic covalent chemistry to prepare an anionic proprotein (A-protein) with abundant phosphate groups. A guanidyl-decorated α-helical polypeptide (LPP) was employed not only to encapsulate A-protein through electrostatic attraction and hydrogen bonding, forming stable nanocomplexes, but also to enhance cell membrane penetration due to its rigid α-helical conformation. Consequently, this strategy mediated the effective delivery of various proteins with different isoelectric points and molecular weights, including α-chymotrypsin, bovine serum albumin, ribonuclease A, cytochrome C, saporin, horseradish peroxidase, β-galactosidase, and anti-phospho-Akt, into cancer cells. More importantly, it enabled efficient delivery of CRISPR-Cas9 ribonucleoproteins to elicit robust polo-like kinase 1 genome editing for inhibiting cancer cell growth. This rationally designed protein delivery system may benefit the development of intracellular protein-based cancer therapy.},
}
MeSH Terms:
show MeSH Terms
hide MeSH Terms
Humans
*Gene Editing
*CRISPR-Cas Systems/genetics
*Peptides/chemistry/pharmacology
Guanidine/chemistry
Cytosol/metabolism
Protein Conformation, alpha-Helical
RevDate: 2025-02-05
CmpDate: 2025-02-05
Phage-mediated intercellular CRISPRi for biocomputation in bacterial consortia.
Nucleic acids research, 53(3):.
Coordinated actions of cells in microbial communities and multicellular organisms enable them to perform complex tasks otherwise difficult for single cells. This has inspired biological engineers to build cellular consortia for larger circuits with improved functionalities while implementing communication systems for coordination among cells. Here, we investigate the signalling dynamics of a phage-mediated synthetic DNA messaging system and couple it with CRISPR interference to build distributed circuits that perform logic gate operations in multicellular bacterial consortia. We find that growth phases of both sender and receiver cells, as well as resource competition between them, shape communication outcomes. Leveraging the easy programmability of DNA messages, we build eight orthogonal signals and demonstrate that intercellular CRISPRi (i-CRISPRi) regulates gene expression across cells. Finally, we multiplex the i-CRISPRi system to implement several multicellular logic gates that involve up to seven cells and take up to three inputs simultaneously, with single- and dual-rail encoding: NOT, YES, AND and AND-AND-NOT. The communication system developed here lays the groundwork for implementing complex biological circuits in engineered bacterial communities, using phage signals for communication.
Additional Links: PMID-39727169
Publisher:
PubMed:
Citation:
show bibtex listing
hide bibtex listing
@article {pmid39727169,
year = {2025},
author = {Pujar, A and Pathania, A and Hopper, C and Pandi, A and Calderón, CR and Függer, M and Nowak, T and Kushwaha, M},
title = {Phage-mediated intercellular CRISPRi for biocomputation in bacterial consortia.},
journal = {Nucleic acids research},
volume = {53},
number = {3},
pages = {},
doi = {10.1093/nar/gkae1256},
pmid = {39727169},
issn = {1362-4962},
support = {//igicosme working group HicDiesMeus/ ; COMBACT//Ile-de-France (IdF) region's DIM-RFSI/ ; BACON//NS2I CNRS/ ; DEPEC MODE//Université Paris-Saclay's STIC department/ ; PHEMO//INRAE's MICA department/ ; ANR-21-CE48-0003//Agence Nationale de la Recherche/ ; },
mesh = {*Bacteriophages/genetics ; *CRISPR-Cas Systems ; Bacteria/genetics/metabolism ; Microbial Consortia/genetics ; Synthetic Biology/methods ; Escherichia coli/genetics/metabolism ; },
abstract = {Coordinated actions of cells in microbial communities and multicellular organisms enable them to perform complex tasks otherwise difficult for single cells. This has inspired biological engineers to build cellular consortia for larger circuits with improved functionalities while implementing communication systems for coordination among cells. Here, we investigate the signalling dynamics of a phage-mediated synthetic DNA messaging system and couple it with CRISPR interference to build distributed circuits that perform logic gate operations in multicellular bacterial consortia. We find that growth phases of both sender and receiver cells, as well as resource competition between them, shape communication outcomes. Leveraging the easy programmability of DNA messages, we build eight orthogonal signals and demonstrate that intercellular CRISPRi (i-CRISPRi) regulates gene expression across cells. Finally, we multiplex the i-CRISPRi system to implement several multicellular logic gates that involve up to seven cells and take up to three inputs simultaneously, with single- and dual-rail encoding: NOT, YES, AND and AND-AND-NOT. The communication system developed here lays the groundwork for implementing complex biological circuits in engineered bacterial communities, using phage signals for communication.},
}
MeSH Terms:
show MeSH Terms
hide MeSH Terms
*Bacteriophages/genetics
*CRISPR-Cas Systems
Bacteria/genetics/metabolism
Microbial Consortia/genetics
Synthetic Biology/methods
Escherichia coli/genetics/metabolism
RevDate: 2025-02-05
CmpDate: 2025-02-05
A modular toolbox for the optogenetic deactivation of transcription.
Nucleic acids research, 53(3):.
Light-controlled transcriptional activation is a commonly used optogenetic strategy that allows researchers to regulate gene expression with high spatiotemporal precision. The vast majority of existing tools are, however, limited to light-triggered induction of gene expression. Here, we inverted this mode of action and created optogenetic systems capable of efficiently terminating transcriptional activation in response to blue light. First, we designed highly compact regulators by photo-controlling the VP16 (pcVP16) transactivation peptide. Then, applying a two-hybrid strategy, we engineered LOOMINA (light off-operated modular inductor of transcriptional activation), a versatile transcriptional control platform for mammalian cells that is compatible with various effector proteins. Leveraging the flexibility of CRISPR systems, we combined LOOMINA with dCas9 to control transcription with blue light from endogenous promoters with exceptionally high dynamic ranges in multiple cell lines. Functionally and mechanistically, the versatile LOOMINA platform and the exceptionally compact pcVP16 transactivator represent valuable additions to the optogenetic repertoire for transcriptional regulation.
Additional Links: PMID-39676667
Publisher:
PubMed:
Citation:
show bibtex listing
hide bibtex listing
@article {pmid39676667,
year = {2025},
author = {Muench, P and Fiumara, M and Southern, N and Coda, D and Aschenbrenner, S and Correia, B and Gräff, J and Niopek, D and Mathony, J},
title = {A modular toolbox for the optogenetic deactivation of transcription.},
journal = {Nucleic acids research},
volume = {53},
number = {3},
pages = {},
doi = {10.1093/nar/gkae1237},
pmid = {39676667},
issn = {1362-4962},
support = {101041570//European Union/ ; 453202693//German Research Foundation/ ; //Executive Agency/ ; //Aventis Foundation/ ; CoG 101043457//ERC/SERI/ ; //Vallee Foundation/ ; //HFSP/ ; 310 030_197752/SNSF_/Swiss National Science Foundation/Switzerland ; CoG 101043457//SERI/ ; },
mesh = {*Optogenetics/methods ; Humans ; *Transcriptional Activation ; HEK293 Cells ; *Light ; Promoter Regions, Genetic ; CRISPR-Cas Systems ; Herpes Simplex Virus Protein Vmw65/genetics/metabolism ; Transcription, Genetic ; Gene Expression Regulation ; Animals ; },
abstract = {Light-controlled transcriptional activation is a commonly used optogenetic strategy that allows researchers to regulate gene expression with high spatiotemporal precision. The vast majority of existing tools are, however, limited to light-triggered induction of gene expression. Here, we inverted this mode of action and created optogenetic systems capable of efficiently terminating transcriptional activation in response to blue light. First, we designed highly compact regulators by photo-controlling the VP16 (pcVP16) transactivation peptide. Then, applying a two-hybrid strategy, we engineered LOOMINA (light off-operated modular inductor of transcriptional activation), a versatile transcriptional control platform for mammalian cells that is compatible with various effector proteins. Leveraging the flexibility of CRISPR systems, we combined LOOMINA with dCas9 to control transcription with blue light from endogenous promoters with exceptionally high dynamic ranges in multiple cell lines. Functionally and mechanistically, the versatile LOOMINA platform and the exceptionally compact pcVP16 transactivator represent valuable additions to the optogenetic repertoire for transcriptional regulation.},
}
MeSH Terms:
show MeSH Terms
hide MeSH Terms
*Optogenetics/methods
Humans
*Transcriptional Activation
HEK293 Cells
*Light
Promoter Regions, Genetic
CRISPR-Cas Systems
Herpes Simplex Virus Protein Vmw65/genetics/metabolism
Transcription, Genetic
Gene Expression Regulation
Animals
RevDate: 2025-02-05
CmpDate: 2025-02-05
The glycosyltransferase ST3GAL4 drives immune evasion in acute myeloid leukemia by synthesizing ligands for the glyco-immune checkpoint receptor Siglec-9.
Leukemia, 39(2):346-359.
Immunotherapy has demonstrated promise as a treatment for acute myeloid leukemia (AML). However, there is still an urgent need to identify new molecules that inhibit the immune response to AML. Most prior research in this area has focused on protein-protein interaction interfaces. While carbohydrates also regulate immune recognition, the role of cell-surface glycans in driving AML immune evasion is comparatively understudied. The Siglecs, for example, are an important family of inhibitory, glycan-binding signaling receptors that have emerged as prime targets for cancer immunotherapy in recent years. In this study, we find that AML cells express ligands for the receptor Siglec-9 at high levels. Integrated CRISPR genomic screening and clinical bioinformatic analysis identified ST3GAL4 as a potential driver of Siglec-9 ligand expression in AML. Depletion of ST3GAL4 by CRISPR-Cas9 knockout (KO) dramatically reduced the expression of Siglec-9 ligands in AML cells. Mass spectrometry analysis of cell-surface glycosylation in ST3GAL4 KO cells revealed that Siglec-9 primarily binds N-linked sialoglycans on these cell types. Finally, we found that ST3GAL4 KO enhanced the sensitivity of AML cells to phagocytosis by Siglec-9-expressing macrophages. This work reveals a novel axis of immune evasion and implicates ST3GAL4 as a possible target for immunotherapy in AML.
Additional Links: PMID-39551873
PubMed:
Citation:
show bibtex listing
hide bibtex listing
@article {pmid39551873,
year = {2025},
author = {Krishnamoorthy, V and Daly, J and Kim, J and Piatnitca, L and Yuen, KA and Kumar, B and Taherzadeh Ghahfarrokhi, M and Bui, TQT and Azadi, P and Vu, LP and Wisnovsky, S},
title = {The glycosyltransferase ST3GAL4 drives immune evasion in acute myeloid leukemia by synthesizing ligands for the glyco-immune checkpoint receptor Siglec-9.},
journal = {Leukemia},
volume = {39},
number = {2},
pages = {346-359},
pmid = {39551873},
issn = {1476-5551},
mesh = {Humans ; *Leukemia, Myeloid, Acute/immunology/metabolism/genetics/pathology ; *Sialyltransferases/metabolism/genetics ; *Antigens, CD/metabolism/genetics ; Ligands ; Immune Evasion ; Sialic Acid Binding Immunoglobulin-like Lectins/metabolism/genetics ; beta-Galactoside alpha-2,3-Sialyltransferase ; Antigens, Differentiation, B-Lymphocyte/metabolism/genetics ; CRISPR-Cas Systems ; Tumor Escape ; },
abstract = {Immunotherapy has demonstrated promise as a treatment for acute myeloid leukemia (AML). However, there is still an urgent need to identify new molecules that inhibit the immune response to AML. Most prior research in this area has focused on protein-protein interaction interfaces. While carbohydrates also regulate immune recognition, the role of cell-surface glycans in driving AML immune evasion is comparatively understudied. The Siglecs, for example, are an important family of inhibitory, glycan-binding signaling receptors that have emerged as prime targets for cancer immunotherapy in recent years. In this study, we find that AML cells express ligands for the receptor Siglec-9 at high levels. Integrated CRISPR genomic screening and clinical bioinformatic analysis identified ST3GAL4 as a potential driver of Siglec-9 ligand expression in AML. Depletion of ST3GAL4 by CRISPR-Cas9 knockout (KO) dramatically reduced the expression of Siglec-9 ligands in AML cells. Mass spectrometry analysis of cell-surface glycosylation in ST3GAL4 KO cells revealed that Siglec-9 primarily binds N-linked sialoglycans on these cell types. Finally, we found that ST3GAL4 KO enhanced the sensitivity of AML cells to phagocytosis by Siglec-9-expressing macrophages. This work reveals a novel axis of immune evasion and implicates ST3GAL4 as a possible target for immunotherapy in AML.},
}
MeSH Terms:
show MeSH Terms
hide MeSH Terms
Humans
*Leukemia, Myeloid, Acute/immunology/metabolism/genetics/pathology
*Sialyltransferases/metabolism/genetics
*Antigens, CD/metabolism/genetics
Ligands
Immune Evasion
Sialic Acid Binding Immunoglobulin-like Lectins/metabolism/genetics
beta-Galactoside alpha-2,3-Sialyltransferase
Antigens, Differentiation, B-Lymphocyte/metabolism/genetics
CRISPR-Cas Systems
Tumor Escape
RevDate: 2025-02-05
CmpDate: 2025-02-05
CRISPR/Cas9-based editing of NF-YC4 promoters yields high-protein rice and soybean.
The New phytologist, 245(5):2103-2116.
Genome editing is a revolution in biotechnology for crop improvement with the final product lacking transgenes. However, most derived traits have been generated through edits that create gene knockouts. Our study pioneers a novel approach, utilizing gene editing to enhance gene expression by eliminating transcriptional repressor binding motifs. Building upon our prior research demonstrating the protein-boosting effects of the transcription factor NF-YC4, we identified conserved motifs targeted by RAV and WRKY repressors in the NF-YC4 promoters from rice (Oryza sativa) and soybean (Glycine max). Leveraging CRISPR/Cas9 technology, we deleted these motifs, resulting in reduced repressor binding and increased NF-YC4 expression. This strategy led to increased protein content and reduced carbohydrate levels in the edited rice and soybean plants, with rice exhibiting up to a 68% increase in leaf protein and a 17% increase in seed protein, and soybean showing up to a 25% increase in leaf protein and an 11% increase in seed protein. Our findings provide a blueprint for enhancing gene expression through precise genomic deletions in noncoding sequences, promising improved agricultural productivity and nutritional quality.
Additional Links: PMID-39307530
Publisher:
PubMed:
Citation:
show bibtex listing
hide bibtex listing
@article {pmid39307530,
year = {2025},
author = {Wang, L and O'Conner, S and Tanvir, R and Zheng, W and Cothron, S and Towery, K and Bi, H and Ellison, EE and Yang, B and Voytas, DF and Li, L},
title = {CRISPR/Cas9-based editing of NF-YC4 promoters yields high-protein rice and soybean.},
journal = {The New phytologist},
volume = {245},
number = {5},
pages = {2103-2116},
doi = {10.1111/nph.20141},
pmid = {39307530},
issn = {1469-8137},
support = {RCZK202468//Shihezi University/ ; //Iowa Soybean Association/ ; //Amfora, Inc./ ; 0951170//National Science Foundation/ ; 2238942//National Science Foundation/ ; CZ001616//Tianchiyingcai Foundation/ ; },
mesh = {*Oryza/genetics ; *Glycine max/genetics ; *CRISPR-Cas Systems ; *Gene Editing/methods ; *Promoter Regions, Genetic/genetics ; *Plant Proteins/genetics/metabolism ; *Gene Expression Regulation, Plant ; Plants, Genetically Modified ; CCAAT-Binding Factor/genetics/metabolism ; Base Sequence ; Plant Leaves/genetics/metabolism ; Seeds/genetics ; },
abstract = {Genome editing is a revolution in biotechnology for crop improvement with the final product lacking transgenes. However, most derived traits have been generated through edits that create gene knockouts. Our study pioneers a novel approach, utilizing gene editing to enhance gene expression by eliminating transcriptional repressor binding motifs. Building upon our prior research demonstrating the protein-boosting effects of the transcription factor NF-YC4, we identified conserved motifs targeted by RAV and WRKY repressors in the NF-YC4 promoters from rice (Oryza sativa) and soybean (Glycine max). Leveraging CRISPR/Cas9 technology, we deleted these motifs, resulting in reduced repressor binding and increased NF-YC4 expression. This strategy led to increased protein content and reduced carbohydrate levels in the edited rice and soybean plants, with rice exhibiting up to a 68% increase in leaf protein and a 17% increase in seed protein, and soybean showing up to a 25% increase in leaf protein and an 11% increase in seed protein. Our findings provide a blueprint for enhancing gene expression through precise genomic deletions in noncoding sequences, promising improved agricultural productivity and nutritional quality.},
}
MeSH Terms:
show MeSH Terms
hide MeSH Terms
*Oryza/genetics
*Glycine max/genetics
*CRISPR-Cas Systems
*Gene Editing/methods
*Promoter Regions, Genetic/genetics
*Plant Proteins/genetics/metabolism
*Gene Expression Regulation, Plant
Plants, Genetically Modified
CCAAT-Binding Factor/genetics/metabolism
Base Sequence
Plant Leaves/genetics/metabolism
Seeds/genetics
RevDate: 2025-02-04
Assessing spacer acquisition rates in E. coli type I-E CRISPR arrays.
Frontiers in microbiology, 15:1498959.
CRISPR/Cas is an adaptive defense mechanism protecting prokaryotes from viruses and other potentially harmful genetic elements. Through an adaptation process, short "spacer" sequences, captured from these elements and incorporated into a CRISPR array, provide target specificity for the immune response. CRISPR arrays and array expansion are also central to many emerging biotechnologies. The rates at which spacers integrate into native arrays within bacterial populations have not been quantified. Here, we measure naïve spacer acquisition rates in Escherichia coli Type I-E CRISPR, identify factors that affect these rates, and model this process fundamental to CRISPR/Cas defense. Prolonged Cas1-Cas2 expression produced fewer new spacers per cell on average than predicted by the model. Subsequent experiments revealed that this was due to a mean fitness reduction linked to array-expanded populations. In addition, the expression of heterologous non-homologous end-joining DNA-repair genes was found to augment spacer acquisition rates, translating to enhanced phage infection defense. Together, these results demonstrate the impact of intracellular factors that modulate spacer acquisition and identify an intrinsic fitness effect associated with array-expanded populations.
Additional Links: PMID-39902289
PubMed:
Citation:
show bibtex listing
hide bibtex listing
@article {pmid39902289,
year = {2024},
author = {Peach, LJ and Zhang, H and Weaver, BP and Boedicker, JQ},
title = {Assessing spacer acquisition rates in E. coli type I-E CRISPR arrays.},
journal = {Frontiers in microbiology},
volume = {15},
number = {},
pages = {1498959},
pmid = {39902289},
issn = {1664-302X},
abstract = {CRISPR/Cas is an adaptive defense mechanism protecting prokaryotes from viruses and other potentially harmful genetic elements. Through an adaptation process, short "spacer" sequences, captured from these elements and incorporated into a CRISPR array, provide target specificity for the immune response. CRISPR arrays and array expansion are also central to many emerging biotechnologies. The rates at which spacers integrate into native arrays within bacterial populations have not been quantified. Here, we measure naïve spacer acquisition rates in Escherichia coli Type I-E CRISPR, identify factors that affect these rates, and model this process fundamental to CRISPR/Cas defense. Prolonged Cas1-Cas2 expression produced fewer new spacers per cell on average than predicted by the model. Subsequent experiments revealed that this was due to a mean fitness reduction linked to array-expanded populations. In addition, the expression of heterologous non-homologous end-joining DNA-repair genes was found to augment spacer acquisition rates, translating to enhanced phage infection defense. Together, these results demonstrate the impact of intracellular factors that modulate spacer acquisition and identify an intrinsic fitness effect associated with array-expanded populations.},
}
RevDate: 2025-02-04
Current biosensing strategies based on in vitro T7 RNA polymerase reaction.
Biotechnology notes (Amsterdam, Netherlands), 6:59-66.
Recently, a unique behavior of T7 RNA polymerase has expanded its functionality as a biosensing platform. Various biosensors utilizing T7 RNA polymerase, combined with fluorescent aptamers, electrochemical probes, or CRISPR/Cas systems, have been developed to detect analytes, including nucleic acids and non-nucleic acid target, with high specificity and low detection limits. Each approach demonstrates unique strengths, such as real-time monitoring and minimal interference, but also presents challenges in stability, cost, and reaction optimization. This review provides an overview of T7 RNA polymerase's role in biosensing technology, highlighting its potential to advance diagnostics and molecular detection in diverse fields.
Additional Links: PMID-39902056
PubMed:
Citation:
show bibtex listing
hide bibtex listing
@article {pmid39902056,
year = {2025},
author = {Sumanto Marpaung, DS and Yap Sinaga, AO and Damayanti, D and Taharuddin, T and Gumaran, S},
title = {Current biosensing strategies based on in vitro T7 RNA polymerase reaction.},
journal = {Biotechnology notes (Amsterdam, Netherlands)},
volume = {6},
number = {},
pages = {59-66},
pmid = {39902056},
issn = {2665-9069},
abstract = {Recently, a unique behavior of T7 RNA polymerase has expanded its functionality as a biosensing platform. Various biosensors utilizing T7 RNA polymerase, combined with fluorescent aptamers, electrochemical probes, or CRISPR/Cas systems, have been developed to detect analytes, including nucleic acids and non-nucleic acid target, with high specificity and low detection limits. Each approach demonstrates unique strengths, such as real-time monitoring and minimal interference, but also presents challenges in stability, cost, and reaction optimization. This review provides an overview of T7 RNA polymerase's role in biosensing technology, highlighting its potential to advance diagnostics and molecular detection in diverse fields.},
}
RevDate: 2025-02-04
CmpDate: 2025-02-04
The generation and evaluation of TKO/hCD55/hTM/hEPCR gene-modified pigs for clinical organ xenotransplantation.
Frontiers in immunology, 15:1488552.
INTRODUCTION: Genetically edited pigs, modified using CRISPR-Cas9 technology, hold promise as potential sources for xenotransplantation. However, the optimal combination of genetic modifications and their expression levels for initial clinical trials remains unclear. This study investigates the generation of TKO/hCD55/hTM/hEPCR (6GE) pigs and evaluates their compatibility with human immune and coagulation systems.
METHODS: The 6GE pigs were generated through iterative genome editing and F1 generation breeding. Genotyping, flow cytometry, and immunohistochemistry confirmed the knockout of GGTA1, CMAH, and B4GALNT2. Expression levels of human genes (hCD55, hTM, hEPCR) were quantified. In vitro assays using aortic endothelial cells (pAECs) from 6GE pigs assessed human serum IgM and IgG binding, complement cytotoxicity, and thrombin-antithrombin (TAT) complex levels. Blood from gene-edited pigs was used for pathophysiological analysis.
RESULTS: Complete knockout of GGTA1, CMAH, and B4GALNT2 was confirmed in 6GE pigs. The expression of hCD55 and hTM was approximately seven and thirteen times higher than in humans, respectively, while hEPCR levels were comparable to those in humans. In vitro, 6GE pAECs showed significantly reduced binding of human IgM and IgG compared to wild-type pAECs (IgG p<0.01, IgM p<0.0001). Similar to TKO/hCD55 pAECs, 6GE pAECs exhibited a substantial reduction in complement-mediated cytotoxicity (p<0.001) compared to TKO pAECs. Co-expression of hTM and hEPCR in 6GE pigs led to a significant decrease in thrombin-antithrombin (TAT) complex levels in co-culture with human whole blood, compared to WT (p<0.0001), TKO (p<0.01), and TKO/hCD55/hTM pigs (p<0.05). Pathophysiological analysis demonstrated excellent compatibility of 6GE pig kidneys and livers with human immune and coagulation systems. However, 6GE pigs showed increased susceptibility to infection compared to other gene-edited pigs, while TKO/hCD55 pigs were considered safe when they were all bred in a general environment.
DISCUSSION: Highly expressing hCD55, along with the co-expression of hEPCR and hTM genes, is expected to effectively reduce human complement cytotoxicity and enhance anticoagulant efficacy in genetically modified pigs. The 6GE pigs exhibited robust compatibility with human physiological and immune systems, fulfilling the criteria for clinical trials. Furthermore, it is imperative to rear donor pigs in pathogen-free (DPF) facilities to mitigate infection risks and prevent the transmission of porcine pathogens to humans.
Additional Links: PMID-39902050
PubMed:
Citation:
show bibtex listing
hide bibtex listing
@article {pmid39902050,
year = {2024},
author = {Huai, G and Wang, Y and Du, J and Cheng, Z and Xie, Y and Zhou, J and Tang, H and Jiang, Y and Xing, X and Deng, S and Pan, D},
title = {The generation and evaluation of TKO/hCD55/hTM/hEPCR gene-modified pigs for clinical organ xenotransplantation.},
journal = {Frontiers in immunology},
volume = {15},
number = {},
pages = {1488552},
pmid = {39902050},
issn = {1664-3224},
mesh = {Animals ; *Transplantation, Heterologous ; Humans ; *Animals, Genetically Modified ; Swine ; *Galactosyltransferases/genetics ; N-Acetylgalactosaminyltransferases/genetics ; Membrane Cofactor Protein/genetics/metabolism ; Thrombomodulin/genetics ; Gene Knockout Techniques ; Gene Editing/methods ; Endothelial Cells/metabolism/immunology ; CRISPR-Cas Systems ; Immunoglobulin M/immunology ; CD55 Antigens/genetics ; Mixed Function Oxygenases ; },
abstract = {INTRODUCTION: Genetically edited pigs, modified using CRISPR-Cas9 technology, hold promise as potential sources for xenotransplantation. However, the optimal combination of genetic modifications and their expression levels for initial clinical trials remains unclear. This study investigates the generation of TKO/hCD55/hTM/hEPCR (6GE) pigs and evaluates their compatibility with human immune and coagulation systems.
METHODS: The 6GE pigs were generated through iterative genome editing and F1 generation breeding. Genotyping, flow cytometry, and immunohistochemistry confirmed the knockout of GGTA1, CMAH, and B4GALNT2. Expression levels of human genes (hCD55, hTM, hEPCR) were quantified. In vitro assays using aortic endothelial cells (pAECs) from 6GE pigs assessed human serum IgM and IgG binding, complement cytotoxicity, and thrombin-antithrombin (TAT) complex levels. Blood from gene-edited pigs was used for pathophysiological analysis.
RESULTS: Complete knockout of GGTA1, CMAH, and B4GALNT2 was confirmed in 6GE pigs. The expression of hCD55 and hTM was approximately seven and thirteen times higher than in humans, respectively, while hEPCR levels were comparable to those in humans. In vitro, 6GE pAECs showed significantly reduced binding of human IgM and IgG compared to wild-type pAECs (IgG p<0.01, IgM p<0.0001). Similar to TKO/hCD55 pAECs, 6GE pAECs exhibited a substantial reduction in complement-mediated cytotoxicity (p<0.001) compared to TKO pAECs. Co-expression of hTM and hEPCR in 6GE pigs led to a significant decrease in thrombin-antithrombin (TAT) complex levels in co-culture with human whole blood, compared to WT (p<0.0001), TKO (p<0.01), and TKO/hCD55/hTM pigs (p<0.05). Pathophysiological analysis demonstrated excellent compatibility of 6GE pig kidneys and livers with human immune and coagulation systems. However, 6GE pigs showed increased susceptibility to infection compared to other gene-edited pigs, while TKO/hCD55 pigs were considered safe when they were all bred in a general environment.
DISCUSSION: Highly expressing hCD55, along with the co-expression of hEPCR and hTM genes, is expected to effectively reduce human complement cytotoxicity and enhance anticoagulant efficacy in genetically modified pigs. The 6GE pigs exhibited robust compatibility with human physiological and immune systems, fulfilling the criteria for clinical trials. Furthermore, it is imperative to rear donor pigs in pathogen-free (DPF) facilities to mitigate infection risks and prevent the transmission of porcine pathogens to humans.},
}
MeSH Terms:
show MeSH Terms
hide MeSH Terms
Animals
*Transplantation, Heterologous
Humans
*Animals, Genetically Modified
Swine
*Galactosyltransferases/genetics
N-Acetylgalactosaminyltransferases/genetics
Membrane Cofactor Protein/genetics/metabolism
Thrombomodulin/genetics
Gene Knockout Techniques
Gene Editing/methods
Endothelial Cells/metabolism/immunology
CRISPR-Cas Systems
Immunoglobulin M/immunology
CD55 Antigens/genetics
Mixed Function Oxygenases
RevDate: 2025-02-04
CmpDate: 2025-02-04
Effects of Electroporation Timing and Cumulus Cell Attachment on In Vitro Development and Genome Editing of Porcine Embryos.
Reproduction in domestic animals = Zuchthygiene, 60(2):e70011.
Pig genome editing using the oviductal nucleic acid delivery (GONAD) method with electroporation would allow the efficient obtention of genetically modified pigs. However, oocytes and zygotes at various stages after ovulation must be targeted, and cumulus cell attachment and mosaic mutations are major obstacles. Therefore, we investigated whether two parameters (electroporation timing and the cumulus cell attachment) influence the effectiveness of multiplex genome editing by electroporation in porcine oocytes or zygotes. Three gRNAs targeting either GGTA1, CMAH or B4GALNT2 were introduced individually into oocytes and zygotes with and without cumulus cells at three different time points, 0 h before in vitro fertilisation (IVF) and 5 h and 10 h after IVF initiation. The introduction of gRNAs into oocytes and zygotes did not significantly affect the rates of blastocyst formation and total mutation of the resulting blastocysts irrespective of cumulus cell attachment and electroporation timing. In conclusion, the electroporation timing and the cumulus cell attachment did not interfere with the efficient delivery of the CRISPR/Cas9 system to the oocytes/zygotes, indicating that porcine genome editing in the oviduct using GONAD method may be possible.
Additional Links: PMID-39901848
Publisher:
PubMed:
Citation:
show bibtex listing
hide bibtex listing
@article {pmid39901848,
year = {2025},
author = {Torigoe, N and Lin, Q and Liu, B and Nakayama, Y and Nakai, A and Nagahara, M and Tanihara, F and Hirata, M and Otoi, T},
title = {Effects of Electroporation Timing and Cumulus Cell Attachment on In Vitro Development and Genome Editing of Porcine Embryos.},
journal = {Reproduction in domestic animals = Zuchthygiene},
volume = {60},
number = {2},
pages = {e70011},
doi = {10.1111/rda.70011},
pmid = {39901848},
issn = {1439-0531},
support = {JP22H02499//Japan Society for the Promotion of Science/ ; JP22K19896//Japan Society for the Promotion of Science/ ; },
mesh = {Animals ; *Cumulus Cells ; *Electroporation/veterinary/methods ; *Gene Editing/methods/veterinary ; Female ; *Oocytes/physiology ; *CRISPR-Cas Systems ; Fertilization in Vitro/veterinary ; Embryonic Development ; Swine ; Zygote ; RNA, Guide, CRISPR-Cas Systems ; Blastocyst/physiology ; Galactosyltransferases/genetics ; Embryo Culture Techniques/veterinary ; },
abstract = {Pig genome editing using the oviductal nucleic acid delivery (GONAD) method with electroporation would allow the efficient obtention of genetically modified pigs. However, oocytes and zygotes at various stages after ovulation must be targeted, and cumulus cell attachment and mosaic mutations are major obstacles. Therefore, we investigated whether two parameters (electroporation timing and the cumulus cell attachment) influence the effectiveness of multiplex genome editing by electroporation in porcine oocytes or zygotes. Three gRNAs targeting either GGTA1, CMAH or B4GALNT2 were introduced individually into oocytes and zygotes with and without cumulus cells at three different time points, 0 h before in vitro fertilisation (IVF) and 5 h and 10 h after IVF initiation. The introduction of gRNAs into oocytes and zygotes did not significantly affect the rates of blastocyst formation and total mutation of the resulting blastocysts irrespective of cumulus cell attachment and electroporation timing. In conclusion, the electroporation timing and the cumulus cell attachment did not interfere with the efficient delivery of the CRISPR/Cas9 system to the oocytes/zygotes, indicating that porcine genome editing in the oviduct using GONAD method may be possible.},
}
MeSH Terms:
show MeSH Terms
hide MeSH Terms
Animals
*Cumulus Cells
*Electroporation/veterinary/methods
*Gene Editing/methods/veterinary
Female
*Oocytes/physiology
*CRISPR-Cas Systems
Fertilization in Vitro/veterinary
Embryonic Development
Swine
Zygote
RNA, Guide, CRISPR-Cas Systems
Blastocyst/physiology
Galactosyltransferases/genetics
Embryo Culture Techniques/veterinary
RevDate: 2025-02-03
CmpDate: 2025-02-04
Engineering a bacterial toxin deaminase from the DYW-family into a novel cytosine base editor for plants and mammalian cells.
Genome biology, 26(1):18.
Base editors are precise editing tools that employ deaminases to modify target DNA bases. The DYW-family of cytosine deaminases is structurally and phylogenetically distinct and might be harnessed for genome editing tools. We report a novel CRISPR/Cas9-cytosine base editor using SsdA, a DYW-like deaminase and bacterial toxin. A G103S mutation in SsdA enhances C-to-T editing efficiency while reducing its toxicity. Truncations result in an extraordinarily small enzyme. The SsdA-base editor efficiently converts C-to-T in rice and barley protoplasts and induces mutations in rice plants and mammalian cells. The engineered SsdA is a highly efficient genome editing tool.
Additional Links: PMID-39901278
PubMed:
Citation:
show bibtex listing
hide bibtex listing
@article {pmid39901278,
year = {2025},
author = {Zhang, D and Parth, F and da Silva, LM and Ha, TC and Schambach, A and Boch, J},
title = {Engineering a bacterial toxin deaminase from the DYW-family into a novel cytosine base editor for plants and mammalian cells.},
journal = {Genome biology},
volume = {26},
number = {1},
pages = {18},
pmid = {39901278},
issn = {1474-760X},
support = {BO 1496/9-1//Deutsche Forschungsgemeinschaft/ ; },
mesh = {*Gene Editing ; *Cytosine/metabolism ; *CRISPR-Cas Systems ; *Oryza/genetics ; Humans ; *Bacterial Toxins/genetics/metabolism ; Cytosine Deaminase/genetics/metabolism ; Animals ; Hordeum/genetics/enzymology ; Protoplasts/metabolism ; HEK293 Cells ; Mutation ; },
abstract = {Base editors are precise editing tools that employ deaminases to modify target DNA bases. The DYW-family of cytosine deaminases is structurally and phylogenetically distinct and might be harnessed for genome editing tools. We report a novel CRISPR/Cas9-cytosine base editor using SsdA, a DYW-like deaminase and bacterial toxin. A G103S mutation in SsdA enhances C-to-T editing efficiency while reducing its toxicity. Truncations result in an extraordinarily small enzyme. The SsdA-base editor efficiently converts C-to-T in rice and barley protoplasts and induces mutations in rice plants and mammalian cells. The engineered SsdA is a highly efficient genome editing tool.},
}
MeSH Terms:
show MeSH Terms
hide MeSH Terms
*Gene Editing
*Cytosine/metabolism
*CRISPR-Cas Systems
*Oryza/genetics
Humans
*Bacterial Toxins/genetics/metabolism
Cytosine Deaminase/genetics/metabolism
Animals
Hordeum/genetics/enzymology
Protoplasts/metabolism
HEK293 Cells
Mutation
RevDate: 2025-02-03
CmpDate: 2025-02-03
Isolation of Enterococcus hirae From Fresh White Yak Milk in Ledu District, Qinghai Province, China: A Comparative Genomic Analysis.
Current microbiology, 82(3):111.
Yak milk is a widely consumed dairy product rich in lactic acid bacteria. Although Enterococcus hirae (E. hirae) is commonly found in dairy products and other foods, there is limited information available on its genetic makeup in yak milk. In the present study, 10 E. hirae strains isolated and identified from fresh white yak milk samples, along with 442 E. hirae strains obtained from the NCBI database (totaling 452 strains), were subjected to comparative genomic analysis. The findings of this study revealed that E. hirae has an open pan-genomic structure that allows for its high adaptability and environmental plasticity. Notably, E. hirae isolates from fresh white yak milk had smaller genomes, encoded more functional genes, and had fewer copies of genes encoding carbohydrate-active enzymes involved in the degradation of oligosaccharide metabolism and autolysin synthesis (CE1, GH73, GH23, and GT4 families) than those from animal and human isolates (P < 0.05). Additionally, fresh white yak milk isolates carried only three intrinsic bacteriocins and lacked virulence factors, CRISPR-Cas systems, and resistance genes linked to pathogenicity, which may be attributed to their specialization in the milk-derived environment. This study provides new insights into the genetic and functional gene diversity of E. hirae and how it adapts to milk-derived habitats.
Additional Links: PMID-39899041
PubMed:
Citation:
show bibtex listing
hide bibtex listing
@article {pmid39899041,
year = {2025},
author = {Lv, H and Sun, J and Guo, Y and Hang, G and Wu, Q and Sun, Z and Zhang, H},
title = {Isolation of Enterococcus hirae From Fresh White Yak Milk in Ledu District, Qinghai Province, China: A Comparative Genomic Analysis.},
journal = {Current microbiology},
volume = {82},
number = {3},
pages = {111},
pmid = {39899041},
issn = {1432-0991},
support = {U22A20540//Natural Science Foundation of China/ ; 2023KYPT0019//Inner Mongolia Science & Technology planning project/ ; BR22-12-03//Basic Research Operating Expenses Program for Colleges and Universities directly under the Inner Mongolia Autonomous Region/ ; },
mesh = {Animals ; *Milk/microbiology ; China ; *Enterococcus hirae/genetics/classification/isolation & purification ; Cattle ; *Genome, Bacterial ; Genomics ; Bacteriocins/genetics ; Virulence Factors/genetics ; Food Microbiology ; Phylogeny ; Humans ; },
abstract = {Yak milk is a widely consumed dairy product rich in lactic acid bacteria. Although Enterococcus hirae (E. hirae) is commonly found in dairy products and other foods, there is limited information available on its genetic makeup in yak milk. In the present study, 10 E. hirae strains isolated and identified from fresh white yak milk samples, along with 442 E. hirae strains obtained from the NCBI database (totaling 452 strains), were subjected to comparative genomic analysis. The findings of this study revealed that E. hirae has an open pan-genomic structure that allows for its high adaptability and environmental plasticity. Notably, E. hirae isolates from fresh white yak milk had smaller genomes, encoded more functional genes, and had fewer copies of genes encoding carbohydrate-active enzymes involved in the degradation of oligosaccharide metabolism and autolysin synthesis (CE1, GH73, GH23, and GT4 families) than those from animal and human isolates (P < 0.05). Additionally, fresh white yak milk isolates carried only three intrinsic bacteriocins and lacked virulence factors, CRISPR-Cas systems, and resistance genes linked to pathogenicity, which may be attributed to their specialization in the milk-derived environment. This study provides new insights into the genetic and functional gene diversity of E. hirae and how it adapts to milk-derived habitats.},
}
MeSH Terms:
show MeSH Terms
hide MeSH Terms
Animals
*Milk/microbiology
China
*Enterococcus hirae/genetics/classification/isolation & purification
Cattle
*Genome, Bacterial
Genomics
Bacteriocins/genetics
Virulence Factors/genetics
Food Microbiology
Phylogeny
Humans
RevDate: 2025-02-03
Characterisation of the CRISPR-Cas systems in Enterococcus faecalis from commercial broiler farm environments and its association with antimicrobial resistance.
British poultry science [Epub ahead of print].
1. Clustered regularly interspaced short palindromic repeats (CRISPR)-associated proteins (Cas) systems have been highlighted for their potential applications in controlling the spread of mobile genetic elements, including antimicrobial resistance (AMR) genes. This study investigated the characteristics of CRISPR-Cas systems in E. faecalis from commercial broiler farms and assessed the impact of these systems on AMR.2. All E. faecalis isolates contained CRISPR2, and CRISPR1-Cas and CRISPR3-Cas were identified in 84 (56.4%) and 144 (96.6%) isolates. A combination of CRISPR2 and CRISPR3-Cas and a combination of CRISPR1-Cas, CRISPR2 and CRISPR3-Cas were each identified in 27 (96.4%) farms.3. There were significant differences between CRISPR-Cas systems for phenotypic AMR: CRISPR1-Cas and CRISPR3-Cas. The E. faecalis isolates without CRISPR1-Cas showed higher resistance to most antimicrobials and had a higher prevalence of multidrug resistance (MDR) than those with CRISPR1-Cas. However, the resistance rate against most antimicrobials and the prevalence of MDR did not differ significantly depending on the presence or absence of CRISPR3-Cas.4. The E. faecalis isolates without CRISPR1-Cas harboured higher levels of all AMR genes, except for tetL, than those with CRISPR1-Cas. However, the E. faecalis isolates with CRISPR3-Cas showed a significant lower prevalence of tetL gene and a significantly higher prevalence of fexA and poxtA genes.5. In the distribution of rep families, the rep9 family was predominant, followed by rep1, rep7, rep2 and rep8 families. Only prevalence of the rep7 family was significantly higher in the E. faecalis isolates without CRISPR1-Cas (15.4%) than in those with CRISPR1-Cas (0%).6. This study is the first report on the characteristics of CRISPR-Cas systems in E. faecalis isolated from commercial broiler farm environments, and the results supported the hypothesis that the development of antimicrobial strategies requires an understanding of the distinctive capabilities between CRISPR1-Cas and CRISPR3-Cas and their underlying resistance mechanisms.
Additional Links: PMID-39899011
Publisher:
PubMed:
Citation:
show bibtex listing
hide bibtex listing
@article {pmid39899011,
year = {2025},
author = {Kim, MB and Lee, YJ},
title = {Characterisation of the CRISPR-Cas systems in Enterococcus faecalis from commercial broiler farm environments and its association with antimicrobial resistance.},
journal = {British poultry science},
volume = {},
number = {},
pages = {1-8},
doi = {10.1080/00071668.2025.2451268},
pmid = {39899011},
issn = {1466-1799},
abstract = {1. Clustered regularly interspaced short palindromic repeats (CRISPR)-associated proteins (Cas) systems have been highlighted for their potential applications in controlling the spread of mobile genetic elements, including antimicrobial resistance (AMR) genes. This study investigated the characteristics of CRISPR-Cas systems in E. faecalis from commercial broiler farms and assessed the impact of these systems on AMR.2. All E. faecalis isolates contained CRISPR2, and CRISPR1-Cas and CRISPR3-Cas were identified in 84 (56.4%) and 144 (96.6%) isolates. A combination of CRISPR2 and CRISPR3-Cas and a combination of CRISPR1-Cas, CRISPR2 and CRISPR3-Cas were each identified in 27 (96.4%) farms.3. There were significant differences between CRISPR-Cas systems for phenotypic AMR: CRISPR1-Cas and CRISPR3-Cas. The E. faecalis isolates without CRISPR1-Cas showed higher resistance to most antimicrobials and had a higher prevalence of multidrug resistance (MDR) than those with CRISPR1-Cas. However, the resistance rate against most antimicrobials and the prevalence of MDR did not differ significantly depending on the presence or absence of CRISPR3-Cas.4. The E. faecalis isolates without CRISPR1-Cas harboured higher levels of all AMR genes, except for tetL, than those with CRISPR1-Cas. However, the E. faecalis isolates with CRISPR3-Cas showed a significant lower prevalence of tetL gene and a significantly higher prevalence of fexA and poxtA genes.5. In the distribution of rep families, the rep9 family was predominant, followed by rep1, rep7, rep2 and rep8 families. Only prevalence of the rep7 family was significantly higher in the E. faecalis isolates without CRISPR1-Cas (15.4%) than in those with CRISPR1-Cas (0%).6. This study is the first report on the characteristics of CRISPR-Cas systems in E. faecalis isolated from commercial broiler farm environments, and the results supported the hypothesis that the development of antimicrobial strategies requires an understanding of the distinctive capabilities between CRISPR1-Cas and CRISPR3-Cas and their underlying resistance mechanisms.},
}
RevDate: 2025-02-04
CmpDate: 2025-02-04
Quantitative Analysis of Phosphorothioate Isomers in CRISPR sgRNA at Single-Residue Resolution Using Endonuclease Digestion Coupled with Liquid Chromatography Cyclic Ion Mobility Mass Spectrometry (LC/cIMS).
Analytical chemistry, 97(4):2223-2231.
Phosphorothioate (PS) modifications in single-guided RNA (sgRNA) are crucial for genome editing applications using the CRISPR/Cas9 system. These modifications may enhance sgRNA stability, pharmacokinetics, and binding to targets, thereby facilitating the desired genetic alterations. Incorporating multiple PS groups at varying positions may introduce chiral centers into the sgRNA backbone, resulting in a complex mixture of constitutional- and stereoisomers that challenges current analytical capabilities for reliable identification and quantification. In this study, we developed an innovative methodology that combines endonuclease digestion of sgRNA with ion pairing reversed-phase liquid chromatography coupled with cyclic ion mobility mass spectrometry (IPRP-LC/cIMS) to fully distinguish PS-induced isomers in a complex mixture. The relative abundance of each isomer was quantified using a two-step method, wherein the ion abundance was sequentially extracted from an LC/MS ion chromatogram and LC/cIMS two-dimensional ion mobiligram. This quantification method was thoroughly evaluated, demonstrating excellent sensitivity, precision, dynamic range, repeatability, and accuracy. In addition, this method enables the investigation of the kinetics of forming PS to phosphodiester (PO) impurities in sgRNA under oxidative stress conditions, offering unprecedented insights into PS stability at a single-residue resolution. In this context, this method highlights the in-depth characterization of PS, demonstrating its capability to support biomedical research, development, and production of sgRNA products.
Additional Links: PMID-39838618
Publisher:
PubMed:
Citation:
show bibtex listing
hide bibtex listing
@article {pmid39838618,
year = {2025},
author = {Su, Y and Zhao, B and Zhang, L and Shen, B and Guo, C and Xiao, H and Li, N},
title = {Quantitative Analysis of Phosphorothioate Isomers in CRISPR sgRNA at Single-Residue Resolution Using Endonuclease Digestion Coupled with Liquid Chromatography Cyclic Ion Mobility Mass Spectrometry (LC/cIMS).},
journal = {Analytical chemistry},
volume = {97},
number = {4},
pages = {2223-2231},
doi = {10.1021/acs.analchem.4c05304},
pmid = {39838618},
issn = {1520-6882},
mesh = {*Mass Spectrometry/methods ; Phosphorothioate Oligonucleotides/chemistry/analysis ; Isomerism ; Ion Mobility Spectrometry/methods ; Chromatography, Liquid/methods ; Endonucleases/metabolism/chemistry ; CRISPR-Cas Systems/genetics ; Chromatography, Reverse-Phase/methods ; },
abstract = {Phosphorothioate (PS) modifications in single-guided RNA (sgRNA) are crucial for genome editing applications using the CRISPR/Cas9 system. These modifications may enhance sgRNA stability, pharmacokinetics, and binding to targets, thereby facilitating the desired genetic alterations. Incorporating multiple PS groups at varying positions may introduce chiral centers into the sgRNA backbone, resulting in a complex mixture of constitutional- and stereoisomers that challenges current analytical capabilities for reliable identification and quantification. In this study, we developed an innovative methodology that combines endonuclease digestion of sgRNA with ion pairing reversed-phase liquid chromatography coupled with cyclic ion mobility mass spectrometry (IPRP-LC/cIMS) to fully distinguish PS-induced isomers in a complex mixture. The relative abundance of each isomer was quantified using a two-step method, wherein the ion abundance was sequentially extracted from an LC/MS ion chromatogram and LC/cIMS two-dimensional ion mobiligram. This quantification method was thoroughly evaluated, demonstrating excellent sensitivity, precision, dynamic range, repeatability, and accuracy. In addition, this method enables the investigation of the kinetics of forming PS to phosphodiester (PO) impurities in sgRNA under oxidative stress conditions, offering unprecedented insights into PS stability at a single-residue resolution. In this context, this method highlights the in-depth characterization of PS, demonstrating its capability to support biomedical research, development, and production of sgRNA products.},
}
MeSH Terms:
show MeSH Terms
hide MeSH Terms
*Mass Spectrometry/methods
Phosphorothioate Oligonucleotides/chemistry/analysis
Isomerism
Ion Mobility Spectrometry/methods
Chromatography, Liquid/methods
Endonucleases/metabolism/chemistry
CRISPR-Cas Systems/genetics
Chromatography, Reverse-Phase/methods
RevDate: 2025-02-04
CmpDate: 2025-02-04
CRISPR-Based Homogeneous Electrochemical Strategy for Near-Zero Background Detection of Breast Cancer Extracellular Vesicles via Fluidity-Enhanced Magnetic Capture Nanoprobe.
Analytical chemistry, 97(4):2176-2185.
Precise identification and analysis of multiple protein biomarkers on the surface of breast cancer cell-derived extracellular vesicles (BC-EVs) are of great significance for noninvasive diagnosis of the breast cancer subtypes, but it remains a major challenge owing to their high heterogeneity and low abundance. Herein, we established a CRISPR-based homogeneous electrochemical strategy for near-zero background and ultrasensitive detection of BC-EVs. To realize the high-performance capture and isolation of BC-EVs, fluidity-enhanced magnetic nanoprobes were facilely prepared. After capturing BC-EVs, the AND logic gate-based catalytic hairpin assembly (CHA) and the trans-cleavage activity of CRISPR-Cas12a against the magnetic signal nanoprobes were triggered successively, generating a significant electrochemical signal. Notably, the as-developed metal-mediated magnetic signal nanoprobes could efficiently decrease the background signal by magnetic separation, endowing the method with a high signal-to-noise ratio. Consequently, by ingeniously integrating DNA logic gate-based CRISPR-CHA signal amplification with dual magnetic nanoprobes in a homogeneous electrochemical strategy, precise identification and ultrasensitive detection of BC-EVs was successfully achieved through simultaneous and specific recognition of dual protein markers on the BC-EVs surface. More importantly, this approach could effectively discriminate specific subgroups of BC-EVs in clinical serum samples, which may provide great opportunities for the accurate diagnosis and prognosis evaluation of breast cancer in a noninvasive manner.
Additional Links: PMID-39829104
Publisher:
PubMed:
Citation:
show bibtex listing
hide bibtex listing
@article {pmid39829104,
year = {2025},
author = {Yang, L and Zhang, J and Zhang, J and Hou, T and Gao, Q and Liu, X and Li, F},
title = {CRISPR-Based Homogeneous Electrochemical Strategy for Near-Zero Background Detection of Breast Cancer Extracellular Vesicles via Fluidity-Enhanced Magnetic Capture Nanoprobe.},
journal = {Analytical chemistry},
volume = {97},
number = {4},
pages = {2176-2185},
doi = {10.1021/acs.analchem.4c05181},
pmid = {39829104},
issn = {1520-6882},
mesh = {Humans ; *Breast Neoplasms/diagnosis ; *Electrochemical Techniques ; *Extracellular Vesicles/chemistry ; Female ; Magnetite Nanoparticles/chemistry ; CRISPR-Cas Systems/genetics ; },
abstract = {Precise identification and analysis of multiple protein biomarkers on the surface of breast cancer cell-derived extracellular vesicles (BC-EVs) are of great significance for noninvasive diagnosis of the breast cancer subtypes, but it remains a major challenge owing to their high heterogeneity and low abundance. Herein, we established a CRISPR-based homogeneous electrochemical strategy for near-zero background and ultrasensitive detection of BC-EVs. To realize the high-performance capture and isolation of BC-EVs, fluidity-enhanced magnetic nanoprobes were facilely prepared. After capturing BC-EVs, the AND logic gate-based catalytic hairpin assembly (CHA) and the trans-cleavage activity of CRISPR-Cas12a against the magnetic signal nanoprobes were triggered successively, generating a significant electrochemical signal. Notably, the as-developed metal-mediated magnetic signal nanoprobes could efficiently decrease the background signal by magnetic separation, endowing the method with a high signal-to-noise ratio. Consequently, by ingeniously integrating DNA logic gate-based CRISPR-CHA signal amplification with dual magnetic nanoprobes in a homogeneous electrochemical strategy, precise identification and ultrasensitive detection of BC-EVs was successfully achieved through simultaneous and specific recognition of dual protein markers on the BC-EVs surface. More importantly, this approach could effectively discriminate specific subgroups of BC-EVs in clinical serum samples, which may provide great opportunities for the accurate diagnosis and prognosis evaluation of breast cancer in a noninvasive manner.},
}
MeSH Terms:
show MeSH Terms
hide MeSH Terms
Humans
*Breast Neoplasms/diagnosis
*Electrochemical Techniques
*Extracellular Vesicles/chemistry
Female
Magnetite Nanoparticles/chemistry
CRISPR-Cas Systems/genetics
RevDate: 2025-02-04
CmpDate: 2025-02-04
CRISPR/Cas9-mediated genomic insertion of functional genes into Lactiplantibacillus plantarum WCFS1.
Microbiology spectrum, 13(2):e0202524.
UNLABELLED: Lactiplantibacillus plantarum, a natural inhabitant of the human body, is a promising candidate vehicle for vaccine delivery. An obstacle in developing bacterial delivery vehicles is generating a production strain that lacks antibiotic resistance genes and contains minimal foreign DNA. To deal with this obstacle, we have constructed a finetuned, inducible two-plasmid CRISPR/Cas9-system for chromosomal gene insertion in L. plantarum. The knock-in plasmid was designed with a cassette-like structure to simplify the insertion of target DNA and streamline the CRISPR/Cas9 genome editing, bringing it one step closer to becoming a routine procedure. We demonstrate that the system enables efficient insertion of expression cassettes for both inducible and constitutive production of a fluorescent reporter protein, mCherry, and for inducible production of the receptor-binding domain (RBD) of the SARS-CoV-2 virus. Two variants of RBD were successfully expressed, one directed to the cytoplasm and one directed to the cell surface. All the knock-in strains produced the target protein, although with lower yields than strains with plasmid-encoded expression.
IMPORTANCE: Genetic engineering of lactic acid bacteria, such as Lactiplantibacillus plantarum, has proven to be difficult. This study presents an inducible two-plasmid CRISPR/Cas9-system for inserting genes into the chromosome of Lactiplantibacillus plantarum. Our system successfully knock-in four expression cassettes varying in length from ~800-1,300 bp with high efficiency and insert an expression cassette encoding a SARS-CoV-2 antigen receptor-binding domain (RBD) with an anchor mediating surface display, which has not been achieved previously using CRISPR/Cas9. We demonstrate the production of the insertion genes. Importantly, the plasmid carrying the SgRNA, Cas9, and homology-directed repair template is designed for easy component exchange. These plasmids represent valuable contributions to the field as they could facilitate rapid CRISPR/Cas9 engineering of L. plantarum strains.
Additional Links: PMID-39817779
Publisher:
PubMed:
Citation:
show bibtex listing
hide bibtex listing
@article {pmid39817779,
year = {2025},
author = {Wiull, K and Haugen, LK and Eijsink, VGH and Mathiesen, G},
title = {CRISPR/Cas9-mediated genomic insertion of functional genes into Lactiplantibacillus plantarum WCFS1.},
journal = {Microbiology spectrum},
volume = {13},
number = {2},
pages = {e0202524},
doi = {10.1128/spectrum.02025-24},
pmid = {39817779},
issn = {2165-0497},
support = {//Norges Miljø- og Biovitenskapelige Universitet (NMBU)/ ; },
mesh = {*CRISPR-Cas Systems ; *Plasmids/genetics ; *Gene Editing/methods ; Lactobacillus plantarum/genetics ; SARS-CoV-2/genetics ; Gene Knock-In Techniques/methods ; Mutagenesis, Insertional ; Humans ; },
abstract = {UNLABELLED: Lactiplantibacillus plantarum, a natural inhabitant of the human body, is a promising candidate vehicle for vaccine delivery. An obstacle in developing bacterial delivery vehicles is generating a production strain that lacks antibiotic resistance genes and contains minimal foreign DNA. To deal with this obstacle, we have constructed a finetuned, inducible two-plasmid CRISPR/Cas9-system for chromosomal gene insertion in L. plantarum. The knock-in plasmid was designed with a cassette-like structure to simplify the insertion of target DNA and streamline the CRISPR/Cas9 genome editing, bringing it one step closer to becoming a routine procedure. We demonstrate that the system enables efficient insertion of expression cassettes for both inducible and constitutive production of a fluorescent reporter protein, mCherry, and for inducible production of the receptor-binding domain (RBD) of the SARS-CoV-2 virus. Two variants of RBD were successfully expressed, one directed to the cytoplasm and one directed to the cell surface. All the knock-in strains produced the target protein, although with lower yields than strains with plasmid-encoded expression.
IMPORTANCE: Genetic engineering of lactic acid bacteria, such as Lactiplantibacillus plantarum, has proven to be difficult. This study presents an inducible two-plasmid CRISPR/Cas9-system for inserting genes into the chromosome of Lactiplantibacillus plantarum. Our system successfully knock-in four expression cassettes varying in length from ~800-1,300 bp with high efficiency and insert an expression cassette encoding a SARS-CoV-2 antigen receptor-binding domain (RBD) with an anchor mediating surface display, which has not been achieved previously using CRISPR/Cas9. We demonstrate the production of the insertion genes. Importantly, the plasmid carrying the SgRNA, Cas9, and homology-directed repair template is designed for easy component exchange. These plasmids represent valuable contributions to the field as they could facilitate rapid CRISPR/Cas9 engineering of L. plantarum strains.},
}
MeSH Terms:
show MeSH Terms
hide MeSH Terms
*CRISPR-Cas Systems
*Plasmids/genetics
*Gene Editing/methods
Lactobacillus plantarum/genetics
SARS-CoV-2/genetics
Gene Knock-In Techniques/methods
Mutagenesis, Insertional
Humans
RevDate: 2025-02-04
CmpDate: 2025-02-04
Optimized husbandry and targeted gene-editing for the cnidarian Nematostella vectensis.
Development (Cambridge, England), 152(2):.
Optimized laboratory conditions for research models are crucial for the success of scientific projects. This includes controlling the entire life cycle, having access to all developmental stages and maintaining stable physiological conditions. Reducing the life cycle of a research model can also enhance the access to biological material and speed up genetic tool development. Thus, we optimized the rearing conditions for the sea anemone Nematostella vectensis, a cnidarian research model, to study embryonic and post-metamorphic processes, such as regeneration. We adopted a semi-automated aquaculture system for N. vectensis and developed a dietary protocol optimized for the different life stages. Thereby, we increased spawning efficiencies, juvenile growth and survival rates, and considerably reduced the overall life cycle down to 2 months. To further improve the obtention of CRISPR-Cas9 mutants, we optimized the design of sgRNAs leading to full knockout animals in F0 polyps using a single sgRNA. Finally, we show that NHEJ-mediated transgene insertion is possible in N. vectensis. In summary, our study provides additional resources for the scientific community that uses or plans to use N. vectensis as a research model.
Additional Links: PMID-39776154
Publisher:
PubMed:
Citation:
show bibtex listing
hide bibtex listing
@article {pmid39776154,
year = {2025},
author = {Carvalho, JE and Burtin, M and Detournay, O and Amiel, AR and Röttinger, E},
title = {Optimized husbandry and targeted gene-editing for the cnidarian Nematostella vectensis.},
journal = {Development (Cambridge, England)},
volume = {152},
number = {2},
pages = {},
doi = {10.1242/dev.204387},
pmid = {39776154},
issn = {1477-9129},
support = {ANR-15-IDEX-01//Agence Nationale de la Recherche/ ; ANR-20-CE13-014//Agence Nationale de la Recherche/ ; SPF20170938703//Fondation pour la Recherche Médicale/ ; SPF20130526781//Fondation pour la Recherche Médicale/ ; N/A//Région SUD/ ; DBM//Institut des sciences biologiques/ ; //Centre national de la Recherche Scientifique/ ; },
mesh = {Animals ; *Sea Anemones/genetics ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; Aquaculture/methods ; Animal Husbandry/methods ; RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; Transgenes ; Animals, Genetically Modified ; },
abstract = {Optimized laboratory conditions for research models are crucial for the success of scientific projects. This includes controlling the entire life cycle, having access to all developmental stages and maintaining stable physiological conditions. Reducing the life cycle of a research model can also enhance the access to biological material and speed up genetic tool development. Thus, we optimized the rearing conditions for the sea anemone Nematostella vectensis, a cnidarian research model, to study embryonic and post-metamorphic processes, such as regeneration. We adopted a semi-automated aquaculture system for N. vectensis and developed a dietary protocol optimized for the different life stages. Thereby, we increased spawning efficiencies, juvenile growth and survival rates, and considerably reduced the overall life cycle down to 2 months. To further improve the obtention of CRISPR-Cas9 mutants, we optimized the design of sgRNAs leading to full knockout animals in F0 polyps using a single sgRNA. Finally, we show that NHEJ-mediated transgene insertion is possible in N. vectensis. In summary, our study provides additional resources for the scientific community that uses or plans to use N. vectensis as a research model.},
}
MeSH Terms:
show MeSH Terms
hide MeSH Terms
Animals
*Sea Anemones/genetics
*Gene Editing/methods
*CRISPR-Cas Systems/genetics
Aquaculture/methods
Animal Husbandry/methods
RNA, Guide, CRISPR-Cas Systems/genetics/metabolism
Transgenes
Animals, Genetically Modified
RevDate: 2025-02-03
Characterization of Rationally Designed CRISPR/Cas9-Based DNA Methyltransferases with Distinct Methyltransferase and Gene Silencing Activities in Human Cell Lines and Primary Human T Cells.
ACS synthetic biology [Epub ahead of print].
Nuclease-deactivated Cas (dCas) proteins can be used to recruit epigenetic effectors, and this class of epigenetic editing technologies has revolutionized the ability to synthetically control the mammalian epigenome and transcriptome. DNA methylation is one of the most important and well-characterized epigenetic modifications in mammals, and while many different forms of dCas-based DNA methyltransferases (dCas-DNMTs) have been developed for programmable DNA methylation, these tools are frequently poorly tolerated and/or lowly expressed in mammalian cell types. Further, the use of dCas-DNMTs has largely been restricted to cell lines, which limits mechanistic insights in karyotypically normal contexts and hampers translational utility in the longer term. Here, we extend previous insights into the rational design of the catalytic core of the mammalian DNMT3A methyltransferase and test three dCas9-DNMT3A/3L variants across different human cell lines and in primary donor-derived human T cells. We find that mutations within the catalytic core of DNMT3A stabilize the expression of dCas9-DNMT3A/3L fusion proteins in Jurkat T cells without sacrificing DNA methylation or gene-silencing performance. We also show that these rationally engineered mutations in DNMT3A alter DNA methylation profiles at loci targeted with dCas9-DNMT3A/3L in cell lines and donor-derived human T cells. Finally, we leverage the transcriptionally repressive effects of dCas9-DNMT3A/3L variants to functionally link the expression of a key immunomodulatory transcription factor to cytokine secretion in donor-derived T cells. Overall, our work expands the synthetic biology toolkit for epigenetic editing and provides a roadmap for the use of engineered dCas-based DNMTs in primary mammalian cell types.
Additional Links: PMID-39898483
Publisher:
PubMed:
Citation:
show bibtex listing
hide bibtex listing
@article {pmid39898483,
year = {2025},
author = {Guerra-Resendez, RS and Lydon, SL and Ma, AJ and Bedford, GC and Reed, DR and Kim, S and Terán, ER and Nishiguchi, T and Escobar, M and DiNardo, AR and Hilton, IB},
title = {Characterization of Rationally Designed CRISPR/Cas9-Based DNA Methyltransferases with Distinct Methyltransferase and Gene Silencing Activities in Human Cell Lines and Primary Human T Cells.},
journal = {ACS synthetic biology},
volume = {},
number = {},
pages = {},
doi = {10.1021/acssynbio.4c00569},
pmid = {39898483},
issn = {2161-5063},
abstract = {Nuclease-deactivated Cas (dCas) proteins can be used to recruit epigenetic effectors, and this class of epigenetic editing technologies has revolutionized the ability to synthetically control the mammalian epigenome and transcriptome. DNA methylation is one of the most important and well-characterized epigenetic modifications in mammals, and while many different forms of dCas-based DNA methyltransferases (dCas-DNMTs) have been developed for programmable DNA methylation, these tools are frequently poorly tolerated and/or lowly expressed in mammalian cell types. Further, the use of dCas-DNMTs has largely been restricted to cell lines, which limits mechanistic insights in karyotypically normal contexts and hampers translational utility in the longer term. Here, we extend previous insights into the rational design of the catalytic core of the mammalian DNMT3A methyltransferase and test three dCas9-DNMT3A/3L variants across different human cell lines and in primary donor-derived human T cells. We find that mutations within the catalytic core of DNMT3A stabilize the expression of dCas9-DNMT3A/3L fusion proteins in Jurkat T cells without sacrificing DNA methylation or gene-silencing performance. We also show that these rationally engineered mutations in DNMT3A alter DNA methylation profiles at loci targeted with dCas9-DNMT3A/3L in cell lines and donor-derived human T cells. Finally, we leverage the transcriptionally repressive effects of dCas9-DNMT3A/3L variants to functionally link the expression of a key immunomodulatory transcription factor to cytokine secretion in donor-derived T cells. Overall, our work expands the synthetic biology toolkit for epigenetic editing and provides a roadmap for the use of engineered dCas-based DNMTs in primary mammalian cell types.},
}
RevDate: 2025-02-03
CmpDate: 2025-02-03
Simultaneous gene editing of both nuclei in a dikaryotic strain of Ganoderma lucidum using Cas9-gRNA ribonucleoprotein.
Journal of microbiology (Seoul, Korea), 63(1):e.2409006.
The presence of multiple nuclei in a common cytoplasm poses a significant challenge to genetic modification in mushrooms. Here, we demonstrate successful gene editing in both nuclei of a dikaryotic strain of Ganoderma lucidum using the Cas9-gRNA ribonucleoprotein complex (RNP). The RNP targeting the pyrG gene was introduced into dikaryotic protoplasts of G. lucidum, resulting in the isolation of 31 mycelial colonies resistant to 5-fluoroorotic acid (5-FOA). Twenty-six of these isolates were confirmed as dikaryotic strains by the presence of two distinct A mating type markers, denoted as A1 and A2. All dikaryons exhibited clamp connections on their mycelial hyphae, while the remaining 5 transformants were monokaryotic. Subsequent sequence analysis of PCR amplicons targeting pyrG revealed that two dikaryons harbored disrupted pyrG in both nuclei (pyrG-/pyrG-), while 10 and 14 displayed pyrG+/pyrG- (A1/A2) and pyrG-/pyrG+ (A1/A2) configurations, respectively. The disruption was achieved through non-homologous end joining repair, involving deletion or insertion of DNA fragments at the site of the double-strand break induced by RNP. Importantly, the nuclei were stable throughout 10 serial transfers over a period of 6 months. These findings highlight the capability of RNP to target genes across multiple nuclei within the same cytoplasm.
Additional Links: PMID-39895071
Publisher:
PubMed:
Citation:
show bibtex listing
hide bibtex listing
@article {pmid39895071,
year = {2025},
author = {Choi, YJ and Eom, H and Nandre, R and Kim, M and Oh, YL and Kim, S and Ro, HS},
title = {Simultaneous gene editing of both nuclei in a dikaryotic strain of Ganoderma lucidum using Cas9-gRNA ribonucleoprotein.},
journal = {Journal of microbiology (Seoul, Korea)},
volume = {63},
number = {1},
pages = {e.2409006},
doi = {10.71150/jm.2409006},
pmid = {39895071},
issn = {1976-3794},
support = {2023R1A 2C1007213//National Research Foundation of Korea/ ; //Rural Development Administration/ ; RS-2024-00322425//New Breeding Technologies Development Program/ ; },
mesh = {*Gene Editing/methods ; *Cell Nucleus/metabolism/genetics ; *Ribonucleoproteins/genetics/metabolism ; *Reishi/genetics/metabolism ; RNA, Guide, CRISPR-Cas Systems/genetics ; CRISPR-Cas Systems ; Fungal Proteins/genetics/metabolism ; CRISPR-Associated Protein 9/genetics/metabolism ; Orotidine-5'-Phosphate Decarboxylase/genetics/metabolism ; Protoplasts/metabolism ; },
abstract = {The presence of multiple nuclei in a common cytoplasm poses a significant challenge to genetic modification in mushrooms. Here, we demonstrate successful gene editing in both nuclei of a dikaryotic strain of Ganoderma lucidum using the Cas9-gRNA ribonucleoprotein complex (RNP). The RNP targeting the pyrG gene was introduced into dikaryotic protoplasts of G. lucidum, resulting in the isolation of 31 mycelial colonies resistant to 5-fluoroorotic acid (5-FOA). Twenty-six of these isolates were confirmed as dikaryotic strains by the presence of two distinct A mating type markers, denoted as A1 and A2. All dikaryons exhibited clamp connections on their mycelial hyphae, while the remaining 5 transformants were monokaryotic. Subsequent sequence analysis of PCR amplicons targeting pyrG revealed that two dikaryons harbored disrupted pyrG in both nuclei (pyrG-/pyrG-), while 10 and 14 displayed pyrG+/pyrG- (A1/A2) and pyrG-/pyrG+ (A1/A2) configurations, respectively. The disruption was achieved through non-homologous end joining repair, involving deletion or insertion of DNA fragments at the site of the double-strand break induced by RNP. Importantly, the nuclei were stable throughout 10 serial transfers over a period of 6 months. These findings highlight the capability of RNP to target genes across multiple nuclei within the same cytoplasm.},
}
MeSH Terms:
show MeSH Terms
hide MeSH Terms
*Gene Editing/methods
*Cell Nucleus/metabolism/genetics
*Ribonucleoproteins/genetics/metabolism
*Reishi/genetics/metabolism
RNA, Guide, CRISPR-Cas Systems/genetics
CRISPR-Cas Systems
Fungal Proteins/genetics/metabolism
CRISPR-Associated Protein 9/genetics/metabolism
Orotidine-5'-Phosphate Decarboxylase/genetics/metabolism
Protoplasts/metabolism
RevDate: 2025-02-02
CmpDate: 2025-02-02
The O-glycosyltransferase C1GALT1 promotes EWSR1::FLI1 expression and is a therapeutic target for Ewing sarcoma.
Nature communications, 16(1):1267.
Ewing sarcoma (ES) is an aggressive bone cancer driven by the oncogenic fusion-protein EWSR1::FLI1, which is not present in normal cells and is therefore an attractive therapeutic target. However, as a transcription factor, EWSR1::FLI1 is considered undruggable. Factors that promote EWSR1::FLI1 expression, and thus whose inhibition would reduce EWSR1::FLI1 protein levels and function, are potential drug targets. Here, using genome-scale CRISPR/Cas9 knockout screening, we identify C1GALT1, a galactosyltransferase required for the biosynthesis of many O-glycoproteins, as a factor that promotes EWSR1::FLI1 expression. We show that C1GALT1 acts by O-glycosylating the pivotal Hedgehog (Hh) signaling component Smoothened (SMO), thereby stabilizing SMO and stimulating the Hh pathway, which we find directly activates EWSR1::FLI1 transcription. Itraconazole, an FDA-approved anti-fungal agent that is known to inhibit C1GALT1, reduces EWSR1::FLI1 levels in ES cell lines and suppresses growth of ES xenografts in mice. Our study reveals a therapeutically targetable mechanism that promotes EWSR1::FLI1 expression and ES tumor growth.
Additional Links: PMID-39894896
PubMed:
Citation:
show bibtex listing
hide bibtex listing
@article {pmid39894896,
year = {2025},
author = {Banday, S and Mishra, AK and Rashid, R and Ye, T and Ali, A and Li, J and Yustein, JT and Kelliher, MA and Zhu, LJ and Deibler, SK and Malonia, SK and Green, MR},
title = {The O-glycosyltransferase C1GALT1 promotes EWSR1::FLI1 expression and is a therapeutic target for Ewing sarcoma.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {1267},
pmid = {39894896},
issn = {2041-1723},
mesh = {Humans ; Animals ; *Galactosyltransferases/genetics/metabolism ; *RNA-Binding Protein EWS/genetics/metabolism ; Cell Line, Tumor ; *Sarcoma, Ewing/genetics/metabolism/pathology/drug therapy ; Mice ; *Oncogene Proteins, Fusion/genetics/metabolism ; *Gene Expression Regulation, Neoplastic ; Proto-Oncogene Protein c-fli-1/metabolism/genetics ; Bone Neoplasms/genetics/pathology/metabolism/drug therapy ; Signal Transduction/drug effects ; Xenograft Model Antitumor Assays ; CRISPR-Cas Systems/genetics ; Hedgehog Proteins/metabolism/genetics ; Female ; },
abstract = {Ewing sarcoma (ES) is an aggressive bone cancer driven by the oncogenic fusion-protein EWSR1::FLI1, which is not present in normal cells and is therefore an attractive therapeutic target. However, as a transcription factor, EWSR1::FLI1 is considered undruggable. Factors that promote EWSR1::FLI1 expression, and thus whose inhibition would reduce EWSR1::FLI1 protein levels and function, are potential drug targets. Here, using genome-scale CRISPR/Cas9 knockout screening, we identify C1GALT1, a galactosyltransferase required for the biosynthesis of many O-glycoproteins, as a factor that promotes EWSR1::FLI1 expression. We show that C1GALT1 acts by O-glycosylating the pivotal Hedgehog (Hh) signaling component Smoothened (SMO), thereby stabilizing SMO and stimulating the Hh pathway, which we find directly activates EWSR1::FLI1 transcription. Itraconazole, an FDA-approved anti-fungal agent that is known to inhibit C1GALT1, reduces EWSR1::FLI1 levels in ES cell lines and suppresses growth of ES xenografts in mice. Our study reveals a therapeutically targetable mechanism that promotes EWSR1::FLI1 expression and ES tumor growth.},
}
MeSH Terms:
show MeSH Terms
hide MeSH Terms
Humans
Animals
*Galactosyltransferases/genetics/metabolism
*RNA-Binding Protein EWS/genetics/metabolism
Cell Line, Tumor
*Sarcoma, Ewing/genetics/metabolism/pathology/drug therapy
Mice
*Oncogene Proteins, Fusion/genetics/metabolism
*Gene Expression Regulation, Neoplastic
Proto-Oncogene Protein c-fli-1/metabolism/genetics
Bone Neoplasms/genetics/pathology/metabolism/drug therapy
Signal Transduction/drug effects
Xenograft Model Antitumor Assays
CRISPR-Cas Systems/genetics
Hedgehog Proteins/metabolism/genetics
Female
RevDate: 2025-02-03
CRISPR integrated biosensors: A new paradigm for cancer detection.
Clinica chimica acta; international journal of clinical chemistry, 569:120179 pii:S0009-8981(25)00058-0 [Epub ahead of print].
Cancer remains one of the leading causes of morbidity and mortality globally, necessitating need for advancements of technologies for early therapeutics. Conventional detection methodologies often lag behind in terms of sensitivity, specificity, and cost-effectiveness, leading to delayed diagnosis and inadequate treatment. The need of advanced diagnostic techniques has considerably increased and led to the development of biosensors. Biosensing technologies offer several advantages over conventional methods hence, overcome limitations and improve diagnostic accuracy. Biosensors, particularly CRISPR-Cas based biosensors have emerged as a revolutionary technology for oncology diagnostics due to their high precision and adaptability. CRISPR-based biosensors provide remarkable precision, sensitivity, multiplexing capabilities, specificity, and rapidness for developing a cost-effective and portable point of care diagnostic device for cancer detection. In this review, we have discussed cancer pathogenicity, assessed the traditional detection techniques, and explored the advancements and advantages of biosensors, particularly CRISPR-based biosensors, in the detection of some major cancer types, namely lung, liver, colorectal, prostate, and cervical cancers. CRISPR-based biosensors represent a significant potential in cancer diagnostics, offering precise, cost-effective, and rapid detection of cancer biomarkers. The integration of CRISPR technology with biosensors holds substantial promise for enhancing early detection and improving patient outcomes in cancer diagnostics.
Additional Links: PMID-39894193
Publisher:
PubMed:
Citation:
show bibtex listing
hide bibtex listing
@article {pmid39894193,
year = {2025},
author = {Saini, A and Dilbaghi, N and Yadav, N},
title = {CRISPR integrated biosensors: A new paradigm for cancer detection.},
journal = {Clinica chimica acta; international journal of clinical chemistry},
volume = {569},
number = {},
pages = {120179},
doi = {10.1016/j.cca.2025.120179},
pmid = {39894193},
issn = {1873-3492},
abstract = {Cancer remains one of the leading causes of morbidity and mortality globally, necessitating need for advancements of technologies for early therapeutics. Conventional detection methodologies often lag behind in terms of sensitivity, specificity, and cost-effectiveness, leading to delayed diagnosis and inadequate treatment. The need of advanced diagnostic techniques has considerably increased and led to the development of biosensors. Biosensing technologies offer several advantages over conventional methods hence, overcome limitations and improve diagnostic accuracy. Biosensors, particularly CRISPR-Cas based biosensors have emerged as a revolutionary technology for oncology diagnostics due to their high precision and adaptability. CRISPR-based biosensors provide remarkable precision, sensitivity, multiplexing capabilities, specificity, and rapidness for developing a cost-effective and portable point of care diagnostic device for cancer detection. In this review, we have discussed cancer pathogenicity, assessed the traditional detection techniques, and explored the advancements and advantages of biosensors, particularly CRISPR-based biosensors, in the detection of some major cancer types, namely lung, liver, colorectal, prostate, and cervical cancers. CRISPR-based biosensors represent a significant potential in cancer diagnostics, offering precise, cost-effective, and rapid detection of cancer biomarkers. The integration of CRISPR technology with biosensors holds substantial promise for enhancing early detection and improving patient outcomes in cancer diagnostics.},
}
RevDate: 2025-02-03
CmpDate: 2025-02-03
A CRISPR-Cas12a-based universal rapid scrub typhus diagnostic method targeting 16S rRNA of Orientia tsutsugamushi.
PLoS neglected tropical diseases, 19(1):e0012826 pii:PNTD-D-24-01350.
Scrub typhus is caused by Orientia tsutsugamushi infection and occurs frequently in an area called the Tsutsugamushi Triangle. Currently, there is no vaccine for O. tsutsugamushi, and its infection is treated with antibiotics such as doxycycline. Scrub typhus responds to effective treatment, and early treatment shortens the course of the disease, reduces mortality, and accelerates recovery. Therefore, it is important to rapidly diagnose O. tsutsugamushi infection to ensure successful outcomes. Here, we developed a CRISPR-Cas12a-based diagnostic method targeting the bacterial 16S rRNA to detect O. tsutsugamushi infection of all known genotypes. To reduce the possibility of contamination and increase field applicability, we designed the one-pot assay system in addition to conventional two-pot assay system. Using this method, we successfully detected up to 100 copies of in vitro transcribed O. tsutsugamushi 16S rRNA within 1 hour under isothermal conditions. In blood samples from patients confirmed to be infected with O. tsutsugamushi by nested PCR, the developed method exhibited a clinical sensitivity of 98% and high specificity. These data demonstrate that the presented method is applicable for the rapid and universal diagnosis of scrub typhus to facilitate timely and appropriate treatment.
Additional Links: PMID-39841710
Publisher:
PubMed:
Citation:
show bibtex listing
hide bibtex listing
@article {pmid39841710,
year = {2025},
author = {Park, BJ and Heo, ST and Kim, M and Yoo, JR and Bae, EJ and Kang, SY and Park, S and Han, KR and Lee, KH and Lee, JM and Lee, H and Song, YJ},
title = {A CRISPR-Cas12a-based universal rapid scrub typhus diagnostic method targeting 16S rRNA of Orientia tsutsugamushi.},
journal = {PLoS neglected tropical diseases},
volume = {19},
number = {1},
pages = {e0012826},
doi = {10.1371/journal.pntd.0012826},
pmid = {39841710},
issn = {1935-2735},
mesh = {*Scrub Typhus/diagnosis/microbiology ; *Orientia tsutsugamushi/genetics/isolation & purification ; *RNA, Ribosomal, 16S/genetics ; Humans ; *CRISPR-Cas Systems ; Sensitivity and Specificity ; Molecular Diagnostic Techniques/methods ; Polymerase Chain Reaction/methods ; },
abstract = {Scrub typhus is caused by Orientia tsutsugamushi infection and occurs frequently in an area called the Tsutsugamushi Triangle. Currently, there is no vaccine for O. tsutsugamushi, and its infection is treated with antibiotics such as doxycycline. Scrub typhus responds to effective treatment, and early treatment shortens the course of the disease, reduces mortality, and accelerates recovery. Therefore, it is important to rapidly diagnose O. tsutsugamushi infection to ensure successful outcomes. Here, we developed a CRISPR-Cas12a-based diagnostic method targeting the bacterial 16S rRNA to detect O. tsutsugamushi infection of all known genotypes. To reduce the possibility of contamination and increase field applicability, we designed the one-pot assay system in addition to conventional two-pot assay system. Using this method, we successfully detected up to 100 copies of in vitro transcribed O. tsutsugamushi 16S rRNA within 1 hour under isothermal conditions. In blood samples from patients confirmed to be infected with O. tsutsugamushi by nested PCR, the developed method exhibited a clinical sensitivity of 98% and high specificity. These data demonstrate that the presented method is applicable for the rapid and universal diagnosis of scrub typhus to facilitate timely and appropriate treatment.},
}
MeSH Terms:
show MeSH Terms
hide MeSH Terms
*Scrub Typhus/diagnosis/microbiology
*Orientia tsutsugamushi/genetics/isolation & purification
*RNA, Ribosomal, 16S/genetics
Humans
*CRISPR-Cas Systems
Sensitivity and Specificity
Molecular Diagnostic Techniques/methods
Polymerase Chain Reaction/methods
RevDate: 2025-02-03
CmpDate: 2025-02-03
Drosophila CG11700 may not affect male fecundity-lifespan tradeoff as previously reported.
Molecular biology and evolution, 42(2):.
Our recent investigations on the function of Drosophila CG11700 and CG32744 (Ubi-p5E) genes using CRISPR/Cas9 deletion technology could not repeat or confirm the results on CG11700 shown in our previous study, which was based on P-element excision assay (Zhan Z, Ding Y, Zhao R, Zhang Y, Yu H, Zhou Q, Yang S, Xiang H, Wang W. Rapid functional divergence of a newly evolved polyubiquitin gene in Drosophila and its role in the trade-off between male fecundity and lifespan. Mol Biol Evol. 2012:29(5):1407-1416. doi:10.1093/molbev/msr299). Here, by CRISPR/Cas9 editing, we generated mutants of CG32744 with the whole gene body fully deleted from the genome, and truncated mutants of CG11700 with N-terminal 103 aa deleted out of its total 301 aa peptide sequence. We carefully conducted the male fecundity assay and found that offsprings of the CG11700 mutant were not significantly more than the wild type, inconsistent with our previous report (Zhan et al. 2012). Meanwhile, we repeated the lifespan assay and did not find that the lifespan of the CG11700 mutant was significantly shorter than the wild type as reported (2012). The new results suggest that the CG11700 gene may not affect male fecundity-lifespan tradeoff as previously reported (Zhan et al. 2012). The new results are thus worthy of reporting to avoid possible misleading by the previous results to the scientific community.
Additional Links: PMID-39817463
Publisher:
PubMed:
Citation:
show bibtex listing
hide bibtex listing
@article {pmid39817463,
year = {2025},
author = {Zuo, D and Liu, W and Zhao, R and Zhu, K and Wang, W and Xiang, H},
title = {Drosophila CG11700 may not affect male fecundity-lifespan tradeoff as previously reported.},
journal = {Molecular biology and evolution},
volume = {42},
number = {2},
pages = {},
doi = {10.1093/molbev/msaf003},
pmid = {39817463},
issn = {1537-1719},
mesh = {Animals ; Male ; *Fertility/genetics ; *Longevity/genetics ; *Drosophila Proteins/genetics ; CRISPR-Cas Systems ; Female ; Drosophila/genetics ; Drosophila melanogaster/genetics ; },
abstract = {Our recent investigations on the function of Drosophila CG11700 and CG32744 (Ubi-p5E) genes using CRISPR/Cas9 deletion technology could not repeat or confirm the results on CG11700 shown in our previous study, which was based on P-element excision assay (Zhan Z, Ding Y, Zhao R, Zhang Y, Yu H, Zhou Q, Yang S, Xiang H, Wang W. Rapid functional divergence of a newly evolved polyubiquitin gene in Drosophila and its role in the trade-off between male fecundity and lifespan. Mol Biol Evol. 2012:29(5):1407-1416. doi:10.1093/molbev/msr299). Here, by CRISPR/Cas9 editing, we generated mutants of CG32744 with the whole gene body fully deleted from the genome, and truncated mutants of CG11700 with N-terminal 103 aa deleted out of its total 301 aa peptide sequence. We carefully conducted the male fecundity assay and found that offsprings of the CG11700 mutant were not significantly more than the wild type, inconsistent with our previous report (Zhan et al. 2012). Meanwhile, we repeated the lifespan assay and did not find that the lifespan of the CG11700 mutant was significantly shorter than the wild type as reported (2012). The new results suggest that the CG11700 gene may not affect male fecundity-lifespan tradeoff as previously reported (Zhan et al. 2012). The new results are thus worthy of reporting to avoid possible misleading by the previous results to the scientific community.},
}
MeSH Terms:
show MeSH Terms
hide MeSH Terms
Animals
Male
*Fertility/genetics
*Longevity/genetics
*Drosophila Proteins/genetics
CRISPR-Cas Systems
Female
Drosophila/genetics
Drosophila melanogaster/genetics
RevDate: 2025-02-03
CmpDate: 2025-02-03
Identification of TUBB3 as an immunotherapy target in lung cancer by genome wide in vivo CRISPR screening.
Neoplasia (New York, N.Y.), 60:101100.
Recent development of immune checkpoint inhibitors has revolutionized cancer immunotherapy. Although these drugs show dramatic effects on a subset of cancer patients, many other tumors are non-responsive and the pathological mechanism of the resistance is largely unknown. To identify genes underlying anti-PD-1 immunotherapy resistance using a systematic approach, we performed an in vivo genome wide CRISPR screening in lung cancer cells. We integrated our results with multi-omics clinical data and performed both in vitro and in vivo assays to evaluate the role of the top candidate in regulating cytotoxic T cell killing. We identified TUBB3 as a potential target to overcome the resistance and enhance the efficacy of anti-PD-1 immunotherapy. TUBB3 expression is upregulated in lung cancer patients, and its higher expression correlates with poorer patients' survival. We found that TUBB3 expression was significantly elevated in the non-responders compared to responders in our patient cohort that received immunotherapies. Importantly, the results of our preclinical experiments showed that inhibition of TUBB3 with a small molecule inhibitor synergized with anti-PD-1 treatment and enhanced tumor cell killing by cytotoxic T cells. Consistently, anti-PD-1 resistant cells showed significantly higher expression of TUBB3; however, TUBB3 inhibition rendered the resistant cells more susceptible to T cell killing. Mechanistic studies revealed that blocking TUBB3 suppressed the expression of PD-L1 through the EMT-related SNAI1 gene. Our results provide a rationale for a novel combination therapy consisting of the TUBB3 inhibition and anti-PD-1 immunotherapy for lung cancer.
Additional Links: PMID-39671912
PubMed:
Citation:
show bibtex listing
hide bibtex listing
@article {pmid39671912,
year = {2025},
author = {Zhao, D and Deshpande, R and Wu, K and Tyagi, A and Sharma, S and Wu, SY and Xing, F and O'Neill, S and Ruiz, J and Lyu, F and Watabe, K},
title = {Identification of TUBB3 as an immunotherapy target in lung cancer by genome wide in vivo CRISPR screening.},
journal = {Neoplasia (New York, N.Y.)},
volume = {60},
number = {},
pages = {101100},
pmid = {39671912},
issn = {1476-5586},
support = {P30 CA012197/CA/NCI NIH HHS/United States ; R01 CA173499/CA/NCI NIH HHS/United States ; R01 CA185650/CA/NCI NIH HHS/United States ; R01 CA205067/CA/NCI NIH HHS/United States ; },
mesh = {Humans ; *Lung Neoplasms/genetics/immunology/drug therapy/therapy/pathology ; *Tubulin/genetics/metabolism/immunology ; Animals ; *Immunotherapy/methods ; Mice ; Cell Line, Tumor ; Gene Expression Regulation, Neoplastic ; CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Immune Checkpoint Inhibitors/pharmacology/therapeutic use ; Xenograft Model Antitumor Assays ; Drug Resistance, Neoplasm/genetics ; T-Lymphocytes, Cytotoxic/immunology ; Disease Models, Animal ; Snail Family Transcription Factors/genetics/metabolism ; },
abstract = {Recent development of immune checkpoint inhibitors has revolutionized cancer immunotherapy. Although these drugs show dramatic effects on a subset of cancer patients, many other tumors are non-responsive and the pathological mechanism of the resistance is largely unknown. To identify genes underlying anti-PD-1 immunotherapy resistance using a systematic approach, we performed an in vivo genome wide CRISPR screening in lung cancer cells. We integrated our results with multi-omics clinical data and performed both in vitro and in vivo assays to evaluate the role of the top candidate in regulating cytotoxic T cell killing. We identified TUBB3 as a potential target to overcome the resistance and enhance the efficacy of anti-PD-1 immunotherapy. TUBB3 expression is upregulated in lung cancer patients, and its higher expression correlates with poorer patients' survival. We found that TUBB3 expression was significantly elevated in the non-responders compared to responders in our patient cohort that received immunotherapies. Importantly, the results of our preclinical experiments showed that inhibition of TUBB3 with a small molecule inhibitor synergized with anti-PD-1 treatment and enhanced tumor cell killing by cytotoxic T cells. Consistently, anti-PD-1 resistant cells showed significantly higher expression of TUBB3; however, TUBB3 inhibition rendered the resistant cells more susceptible to T cell killing. Mechanistic studies revealed that blocking TUBB3 suppressed the expression of PD-L1 through the EMT-related SNAI1 gene. Our results provide a rationale for a novel combination therapy consisting of the TUBB3 inhibition and anti-PD-1 immunotherapy for lung cancer.},
}
MeSH Terms:
show MeSH Terms
hide MeSH Terms
Humans
*Lung Neoplasms/genetics/immunology/drug therapy/therapy/pathology
*Tubulin/genetics/metabolism/immunology
Animals
*Immunotherapy/methods
Mice
Cell Line, Tumor
Gene Expression Regulation, Neoplastic
CRISPR-Cas Systems
Clustered Regularly Interspaced Short Palindromic Repeats/genetics
Immune Checkpoint Inhibitors/pharmacology/therapeutic use
Xenograft Model Antitumor Assays
Drug Resistance, Neoplasm/genetics
T-Lymphocytes, Cytotoxic/immunology
Disease Models, Animal
Snail Family Transcription Factors/genetics/metabolism
RevDate: 2025-02-03
CmpDate: 2025-02-03
qTAG: an adaptable plasmid scaffold for CRISPR-based endogenous tagging.
The EMBO journal, 44(3):947-974.
Endogenous tagging enables the study of proteins within their native regulatory context, typically using CRISPR to insert tag sequences directly into the gene sequence. Here, we introduce qTAG, a collection of repair cassettes that makes endogenous tagging more accessible. The cassettes support N- and C-terminal tagging with commonly used selectable markers and feature restriction sites for easy modification. Lox sites also enable the removal of the marker gene after successful integration. We demonstrate the utility of qTAG with a range of diverse tags for applications in fluorescence imaging, proximity labeling, epitope tagging, and targeted protein degradation. The system includes novel tags like mStayGold, offering enhanced brightness and photostability for live-cell imaging of native protein dynamics. Additionally, we explore alternative cassette designs for conditional expression tagging, selectable knockout tagging, and safe-harbor expression. The plasmid collection is available through Addgene, featuring ready-to-use constructs for common subcellular markers and tagging cassettes to target genes of interest. The qTAG system will serve as an open resource for researchers to adapt and tailor their own experiments.
Additional Links: PMID-39668248
PubMed:
Citation:
show bibtex listing
hide bibtex listing
@article {pmid39668248,
year = {2025},
author = {Philip, R and Sharma, A and Matellan, L and Erpf, AC and Hsu, WH and Tkach, JM and Wyatt, HDM and Pelletier, L},
title = {qTAG: an adaptable plasmid scaffold for CRISPR-based endogenous tagging.},
journal = {The EMBO journal},
volume = {44},
number = {3},
pages = {947-974},
pmid = {39668248},
issn = {1460-2075},
support = {187836//Canadian Government | CIHR | Institute of Cancer Research (IC)/ ; 181763//Canadian Government | CIHR | Institute of Cancer Research (IC)/ ; 156297//Canadian Government | Canadian Institutes of Health Research (CIHR)/ ; 167279//Canadian Government | Canadian Institutes of Health Research (CIHR)/ ; N/A//Krembil Foundation/ ; },
mesh = {*Plasmids/genetics/metabolism ; Humans ; *CRISPR-Cas Systems ; HEK293 Cells ; Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {Endogenous tagging enables the study of proteins within their native regulatory context, typically using CRISPR to insert tag sequences directly into the gene sequence. Here, we introduce qTAG, a collection of repair cassettes that makes endogenous tagging more accessible. The cassettes support N- and C-terminal tagging with commonly used selectable markers and feature restriction sites for easy modification. Lox sites also enable the removal of the marker gene after successful integration. We demonstrate the utility of qTAG with a range of diverse tags for applications in fluorescence imaging, proximity labeling, epitope tagging, and targeted protein degradation. The system includes novel tags like mStayGold, offering enhanced brightness and photostability for live-cell imaging of native protein dynamics. Additionally, we explore alternative cassette designs for conditional expression tagging, selectable knockout tagging, and safe-harbor expression. The plasmid collection is available through Addgene, featuring ready-to-use constructs for common subcellular markers and tagging cassettes to target genes of interest. The qTAG system will serve as an open resource for researchers to adapt and tailor their own experiments.},
}
MeSH Terms:
show MeSH Terms
hide MeSH Terms
*Plasmids/genetics/metabolism
Humans
*CRISPR-Cas Systems
HEK293 Cells
Clustered Regularly Interspaced Short Palindromic Repeats
RevDate: 2025-02-01
Efficient genome editing in dicot plants using calreticulin promoter-driven CRISPR/Cas system.
Molecular horticulture, 5(1):9.
Additional Links: PMID-39893465
PubMed:
Citation:
show bibtex listing
hide bibtex listing
@article {pmid39893465,
year = {2025},
author = {Li, B and Shang, Y and Wang, L and Lv, J and Wu, Q and Wang, F and Chao, J and Mao, J and Ding, A and Wu, X and Xue, K and Chen, C and Cui, M and Sun, Y and Zhang, H and Dai, C},
title = {Efficient genome editing in dicot plants using calreticulin promoter-driven CRISPR/Cas system.},
journal = {Molecular horticulture},
volume = {5},
number = {1},
pages = {9},
pmid = {39893465},
issn = {2730-9401},
}
RevDate: 2025-02-01
CmpDate: 2025-02-01
A universal and wide-range cytosine base editor via domain-inlaid and fidelity-optimized CRISPR-FrCas9.
Nature communications, 16(1):1260.
CRISPR-based base editor (BE) offer diverse editing options for genetic engineering of microorganisms, but its application is limited by protospacer adjacent motif (PAM) sequences, context preference, editing window, and off-target effects. Here, a series of iteratively improved cytosine base editors (CBEs) are constructed using the FrCas9 nickase (FrCas9n) with the unique PAM palindromic structure (NNTA) to alleviate these challenges. The deaminase domain-inlaid FrCas9n exhibits an editing range covering 38 nucleotides upstream and downstream of the palindromic PAM, without context preference, which is 6.3 times larger than that of traditional CBEs. Additionally, lower off-target editing is achieved when incorporating high-fidelity mutations at R61A and Q964A in FrCas9n, while maintaining high editing efficiency. The final CBE, HF-ID824-evoCDA-FrCas9n demonstrates broad applicability across different microbes such as Escherichia coli MG1655, Shewanella oneidensis MR-1, and Pseudomonas aeruginosa PAO1. Collectively, this tool offers robust gene editing for facilitating mechanistic studies, functional exploration, and protein evolution in microbes.
Additional Links: PMID-39893181
PubMed:
Citation:
show bibtex listing
hide bibtex listing
@article {pmid39893181,
year = {2025},
author = {Hu, L and Han, J and Wang, HD and Cheng, ZH and Lv, CC and Liu, DF and Yu, HQ},
title = {A universal and wide-range cytosine base editor via domain-inlaid and fidelity-optimized CRISPR-FrCas9.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {1260},
pmid = {39893181},
issn = {2041-1723},
support = {52322002//National Natural Science Foundation of China (National Science Foundation of China)/ ; },
mesh = {*Cytosine/metabolism ; *Gene Editing/methods ; *CRISPR-Cas Systems ; *Escherichia coli/genetics/metabolism ; Pseudomonas aeruginosa/genetics ; Protein Domains ; Deoxyribonuclease I/metabolism/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Mutation ; CRISPR-Associated Protein 9/metabolism/genetics ; },
abstract = {CRISPR-based base editor (BE) offer diverse editing options for genetic engineering of microorganisms, but its application is limited by protospacer adjacent motif (PAM) sequences, context preference, editing window, and off-target effects. Here, a series of iteratively improved cytosine base editors (CBEs) are constructed using the FrCas9 nickase (FrCas9n) with the unique PAM palindromic structure (NNTA) to alleviate these challenges. The deaminase domain-inlaid FrCas9n exhibits an editing range covering 38 nucleotides upstream and downstream of the palindromic PAM, without context preference, which is 6.3 times larger than that of traditional CBEs. Additionally, lower off-target editing is achieved when incorporating high-fidelity mutations at R61A and Q964A in FrCas9n, while maintaining high editing efficiency. The final CBE, HF-ID824-evoCDA-FrCas9n demonstrates broad applicability across different microbes such as Escherichia coli MG1655, Shewanella oneidensis MR-1, and Pseudomonas aeruginosa PAO1. Collectively, this tool offers robust gene editing for facilitating mechanistic studies, functional exploration, and protein evolution in microbes.},
}
MeSH Terms:
show MeSH Terms
hide MeSH Terms
*Cytosine/metabolism
*Gene Editing/methods
*CRISPR-Cas Systems
*Escherichia coli/genetics/metabolism
Pseudomonas aeruginosa/genetics
Protein Domains
Deoxyribonuclease I/metabolism/genetics
Clustered Regularly Interspaced Short Palindromic Repeats/genetics
Mutation
CRISPR-Associated Protein 9/metabolism/genetics
RevDate: 2025-02-01
CmpDate: 2025-02-01
Advances in lignocellulosic feedstocks for bioenergy and bioproducts.
Nature communications, 16(1):1244.
Lignocellulose, an abundant renewable resource, presents a promising alternative for sustainable energy and industrial applications. However, large-scale adoption of lignocellulosic feedstocks faces considerable obstacles, including scalability, bioprocessing efficiency, and resilience to climate change. This Review examines current efforts and future opportunities for leveraging lignocellulosic feedstocks in bio-based energy and products, with a focus on enhancing conversion efficiency and scalability. It also explores emerging biotechnologies such as CRISPR-based genome editing informed by machine learning, aimed at improving feedstock traits and reducing the environmental impact of fossil fuel dependence.
Additional Links: PMID-39893176
PubMed:
Citation:
show bibtex listing
hide bibtex listing
@article {pmid39893176,
year = {2025},
author = {Sulis, DB and Lavoine, N and Sederoff, H and Jiang, X and Marques, BM and Lan, K and Cofre-Vega, C and Barrangou, R and Wang, JP},
title = {Advances in lignocellulosic feedstocks for bioenergy and bioproducts.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {1244},
pmid = {39893176},
issn = {2041-1723},
mesh = {*Lignin/metabolism ; *Biofuels ; *Gene Editing ; Biotechnology/methods ; Biomass ; CRISPR-Cas Systems ; },
abstract = {Lignocellulose, an abundant renewable resource, presents a promising alternative for sustainable energy and industrial applications. However, large-scale adoption of lignocellulosic feedstocks faces considerable obstacles, including scalability, bioprocessing efficiency, and resilience to climate change. This Review examines current efforts and future opportunities for leveraging lignocellulosic feedstocks in bio-based energy and products, with a focus on enhancing conversion efficiency and scalability. It also explores emerging biotechnologies such as CRISPR-based genome editing informed by machine learning, aimed at improving feedstock traits and reducing the environmental impact of fossil fuel dependence.},
}
MeSH Terms:
show MeSH Terms
hide MeSH Terms
*Lignin/metabolism
*Biofuels
*Gene Editing
Biotechnology/methods
Biomass
CRISPR-Cas Systems
RevDate: 2025-02-01
CmpDate: 2025-02-01
Kinome-Focused CRISPR-Cas9 Screens in African Ancestry Patient-Derived Breast Cancer Organoids Identify Essential Kinases and Synergy of EGFR and FGFR1 Inhibition.
Cancer research, 85(3):551-566.
Precision medicine approaches to cancer treatment aim to exploit genomic alterations that are specific to individual patients to tailor therapeutic strategies. Yet, some targetable genes and pathways are essential for tumor cell viability even in the absence of direct genomic alterations. In underrepresented populations, the mutational landscape and determinants of response to existing therapies are poorly characterized because of limited inclusion in clinical trials and studies. One way to reveal tumor essential genes is with genetic screens. Most screens are conducted on cell lines that bear little resemblance to patient tumors, after years of culture under nonphysiologic conditions. To address this problem, we aimed to develop a CRISPR screening pipeline in three-dimensionally grown patient-derived tumor organoid (PDTO) models. A breast cancer PDTO biobank that focused on underrepresented populations, including West African patients, was established and used to conduct a negative-selection kinome-focused CRISPR screen to identify kinases essential for organoid growth and potential targets for combination therapy with EGFR or MEK inhibitors. The screen identified several previously unidentified kinase targets, and the combination of FGFR1 and EGFR inhibitors synergized to block organoid proliferation. Together, these data demonstrate the feasibility of CRISPR-based genetic screens in patient-derived tumor models, including PDTOs from underrepresented patients with cancer, and identify targets for cancer therapy. Significance: Generation of a breast cancer patient-derived tumor organoid biobank focused on underrepresented populations enabled kinome-focused CRISPR screening that identified essential kinases and potential targets for combination therapy with EGFR or MEK inhibitors. See related commentary by Trembath and Spanheimer, p. 407.
Additional Links: PMID-39891928
Publisher:
PubMed:
Citation:
show bibtex listing
hide bibtex listing
@article {pmid39891928,
year = {2025},
author = {Madorsky Rowdo, FP and Martini, R and Ackermann, SE and Tang, CP and Tranquille, M and Irizarry, A and Us, I and Alawa, O and Moyer, JE and Sigouros, M and Nguyen, J and Al Assaad, M and Cheng, E and Ginter, PS and Manohar, J and Stonaker, B and Boateng, R and Oppong, JK and Adjei, EK and Awuah, B and Kyei, I and Aitpillah, FS and Adinku, MO and Ankomah, K and Osei-Bonsu, EB and Gyan, KK and Hoda, S and Newman, L and Mosquera, JM and Sboner, A and Elemento, O and Dow, LE and Davis, MB and Martin, ML},
title = {Kinome-Focused CRISPR-Cas9 Screens in African Ancestry Patient-Derived Breast Cancer Organoids Identify Essential Kinases and Synergy of EGFR and FGFR1 Inhibition.},
journal = {Cancer research},
volume = {85},
number = {3},
pages = {551-566},
doi = {10.1158/0008-5472.CAN-24-0775},
pmid = {39891928},
issn = {1538-7445},
support = {//Weill Cornell Medicine (WCM)/ ; BCRF-22-191//Breast Cancer Research Foundation (BCRF)/ ; CA259396-01//National Cancer Institute (NCI)/ ; },
mesh = {Humans ; *Organoids/pathology/metabolism ; *Breast Neoplasms/genetics/pathology/drug therapy ; Female ; *CRISPR-Cas Systems ; *Receptor, Fibroblast Growth Factor, Type 1/genetics/antagonists & inhibitors/metabolism ; *ErbB Receptors/genetics/antagonists & inhibitors/metabolism ; Protein Kinase Inhibitors/pharmacology ; Black People/genetics ; },
abstract = {Precision medicine approaches to cancer treatment aim to exploit genomic alterations that are specific to individual patients to tailor therapeutic strategies. Yet, some targetable genes and pathways are essential for tumor cell viability even in the absence of direct genomic alterations. In underrepresented populations, the mutational landscape and determinants of response to existing therapies are poorly characterized because of limited inclusion in clinical trials and studies. One way to reveal tumor essential genes is with genetic screens. Most screens are conducted on cell lines that bear little resemblance to patient tumors, after years of culture under nonphysiologic conditions. To address this problem, we aimed to develop a CRISPR screening pipeline in three-dimensionally grown patient-derived tumor organoid (PDTO) models. A breast cancer PDTO biobank that focused on underrepresented populations, including West African patients, was established and used to conduct a negative-selection kinome-focused CRISPR screen to identify kinases essential for organoid growth and potential targets for combination therapy with EGFR or MEK inhibitors. The screen identified several previously unidentified kinase targets, and the combination of FGFR1 and EGFR inhibitors synergized to block organoid proliferation. Together, these data demonstrate the feasibility of CRISPR-based genetic screens in patient-derived tumor models, including PDTOs from underrepresented patients with cancer, and identify targets for cancer therapy. Significance: Generation of a breast cancer patient-derived tumor organoid biobank focused on underrepresented populations enabled kinome-focused CRISPR screening that identified essential kinases and potential targets for combination therapy with EGFR or MEK inhibitors. See related commentary by Trembath and Spanheimer, p. 407.},
}
MeSH Terms:
show MeSH Terms
hide MeSH Terms
Humans
*Organoids/pathology/metabolism
*Breast Neoplasms/genetics/pathology/drug therapy
Female
*CRISPR-Cas Systems
*Receptor, Fibroblast Growth Factor, Type 1/genetics/antagonists & inhibitors/metabolism
*ErbB Receptors/genetics/antagonists & inhibitors/metabolism
Protein Kinase Inhibitors/pharmacology
Black People/genetics
RevDate: 2025-02-01
CmpDate: 2025-02-01
In Search of Representative Translational Cancer Model Systems.
Cancer research, 85(3):407-409.
Racial disparities in cancer outcomes are well documented across tumor types. For patients with breast cancer, Black women are more likely to present with more aggressive molecular features and more likely to die from disease, even after accounting for those features. Recent efforts have been aimed at developing translational model systems for precision medicine strategies, and a major focus has been on patient-derived organoids. Organoids allow for robust in vitro experimental platforms, including drug and CRISPR screens while maintaining more complex cancer and tumor microenvironment subpopulations than cell lines. For results that are broadly translationally relevant, it is important that cancer models are derived from the spectrum of human disease and humans with disease. In this issue of Cancer Research, Madorsky Rowdo and colleagues derive breast cancer organoids from patients with African ancestry and use CRISPR-Cas9 screens to identify novel therapeutic vulnerabilities. These findings demonstrate the promise of representative cancer model systems to facilitate discoveries that are most likely to translate to improved therapy for all patients. See related article by Madorsky Rowdo et al., p. 551.
Additional Links: PMID-39891927
Publisher:
PubMed:
Citation:
show bibtex listing
hide bibtex listing
@article {pmid39891927,
year = {2025},
author = {Trembath, HE and Spanheimer, PM},
title = {In Search of Representative Translational Cancer Model Systems.},
journal = {Cancer research},
volume = {85},
number = {3},
pages = {407-409},
doi = {10.1158/0008-5472.CAN-24-3879},
pmid = {39891927},
issn = {1538-7445},
mesh = {Humans ; *Organoids/pathology ; Female ; Breast Neoplasms/genetics/pathology/therapy ; CRISPR-Cas Systems ; Precision Medicine/methods ; Translational Research, Biomedical/methods ; Neoplasms/genetics/pathology/therapy ; Tumor Microenvironment ; },
abstract = {Racial disparities in cancer outcomes are well documented across tumor types. For patients with breast cancer, Black women are more likely to present with more aggressive molecular features and more likely to die from disease, even after accounting for those features. Recent efforts have been aimed at developing translational model systems for precision medicine strategies, and a major focus has been on patient-derived organoids. Organoids allow for robust in vitro experimental platforms, including drug and CRISPR screens while maintaining more complex cancer and tumor microenvironment subpopulations than cell lines. For results that are broadly translationally relevant, it is important that cancer models are derived from the spectrum of human disease and humans with disease. In this issue of Cancer Research, Madorsky Rowdo and colleagues derive breast cancer organoids from patients with African ancestry and use CRISPR-Cas9 screens to identify novel therapeutic vulnerabilities. These findings demonstrate the promise of representative cancer model systems to facilitate discoveries that are most likely to translate to improved therapy for all patients. See related article by Madorsky Rowdo et al., p. 551.},
}
MeSH Terms:
show MeSH Terms
hide MeSH Terms
Humans
*Organoids/pathology
Female
Breast Neoplasms/genetics/pathology/therapy
CRISPR-Cas Systems
Precision Medicine/methods
Translational Research, Biomedical/methods
Neoplasms/genetics/pathology/therapy
Tumor Microenvironment
RevDate: 2025-02-01
CmpDate: 2025-02-01
Advancing vegetable genetics with gene editing: a pathway to food security and nutritional resilience in climate-shifted environments.
Functional & integrative genomics, 25(1):31.
As global populations grow and climate change increasingly disrupts agricultural systems, ensuring food security and nutritional resilience has become a critical challenge. In addition to grains and legumes, vegetables are very important for both human and animals because they contain vitamins, minerals, and fibre. Enhancing the ability of vegetables to withstand climate change threats is essential; however, traditional breeding methods face challenges due to the complexity of the genomic clonal multiplication process. In the postgenomic era, gene editing (GE) has emerged as a powerful tool for improving vegetables. GE can help to increase traits such as abiotic stress tolerance, herbicide tolerance, and disease resistance; improve agricultural productivity; and improve nutritional content and shelf-life by fine-tuning key genes. GE technologies such as Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein 9 (CRISPR-Cas9) have revolutionized vegetable breeding by enabling specific gene modifications in the genome. This review highlights recent advances in CRISPR-mediated editing across various vegetable species, highlighting successful modifications that increase their resilience to climatic stressors. Additionally, it explores the potential of GE to address malnutrition by increasing the nutrient content of vegetable crops, thereby contributing to public health and food system sustainability. Additionally, it addresses the implementation of GE-guided breeding strategies in agriculture, considering regulatory, ethical, and public acceptance issues. Enhancing vegetable genetics via GE may provide a reliable and nutritious food supply for an expanding global population under more unpredictable environmental circumstances.
Additional Links: PMID-39891757
PubMed:
Citation:
show bibtex listing
hide bibtex listing
@article {pmid39891757,
year = {2025},
author = {Roychowdhury, R and Das, SP and Das, S and Biswas, S and Patel, MK and Kumar, A and Sarker, U and Choudhary, SP and Das, R and Yogendra, K and Gangurde, SS},
title = {Advancing vegetable genetics with gene editing: a pathway to food security and nutritional resilience in climate-shifted environments.},
journal = {Functional & integrative genomics},
volume = {25},
number = {1},
pages = {31},
pmid = {39891757},
issn = {1438-7948},
mesh = {*Gene Editing ; *Food Security ; *Climate Change ; *Vegetables/metabolism/genetics ; *CRISPR-Cas Systems ; Plant Breeding/methods ; Crops, Agricultural/genetics ; Humans ; },
abstract = {As global populations grow and climate change increasingly disrupts agricultural systems, ensuring food security and nutritional resilience has become a critical challenge. In addition to grains and legumes, vegetables are very important for both human and animals because they contain vitamins, minerals, and fibre. Enhancing the ability of vegetables to withstand climate change threats is essential; however, traditional breeding methods face challenges due to the complexity of the genomic clonal multiplication process. In the postgenomic era, gene editing (GE) has emerged as a powerful tool for improving vegetables. GE can help to increase traits such as abiotic stress tolerance, herbicide tolerance, and disease resistance; improve agricultural productivity; and improve nutritional content and shelf-life by fine-tuning key genes. GE technologies such as Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein 9 (CRISPR-Cas9) have revolutionized vegetable breeding by enabling specific gene modifications in the genome. This review highlights recent advances in CRISPR-mediated editing across various vegetable species, highlighting successful modifications that increase their resilience to climatic stressors. Additionally, it explores the potential of GE to address malnutrition by increasing the nutrient content of vegetable crops, thereby contributing to public health and food system sustainability. Additionally, it addresses the implementation of GE-guided breeding strategies in agriculture, considering regulatory, ethical, and public acceptance issues. Enhancing vegetable genetics via GE may provide a reliable and nutritious food supply for an expanding global population under more unpredictable environmental circumstances.},
}
MeSH Terms:
show MeSH Terms
hide MeSH Terms
*Gene Editing
*Food Security
*Climate Change
*Vegetables/metabolism/genetics
*CRISPR-Cas Systems
Plant Breeding/methods
Crops, Agricultural/genetics
Humans
RevDate: 2025-01-31
CmpDate: 2025-01-31
Room temperature CRISPR diagnostics for low-resource settings.
Scientific reports, 15(1):3909.
Maintaining elevated reaction temperatures and multi-step sample preparations increases the costs and complexity of diagnostics, impeding their deployment in low-resource settings. Here, we develop a one-pot, room temperature recombinase polymerase amplification (RPA)-CRISPR reaction that removes these critical challenges. We show that RPA amplification is reduced by several orders of magnitude at 25 °C as compared to 37 °C. Similarly, when coupled to RPA, the performance of multiple Cas12a orthologs, including the widely used LbCas12a, is severely compromised at temperatures below 37 °C. To mitigate these limitations, we identify the ortholog TsCas12a as a highly active nuclease at 25 °C and develop a single-protocol RPA-Cas12a detection reaction with this enzyme. A quantitative kinetic analysis reveals that fast nuclease activation is more critical than higher steady-state trans-cleavage activity for room temperature diagnostic applications. RPA-TsCas12a reactions performed at 25 °C effectively detected HPV-16 in crudely prepared cervical swab samples with high sensitivity and specificity using both optical and lateral flow readouts. The reactions developed herein reduce the complexity and equipment requirements for affordable diagnostics in low- and middle-income countries.
Additional Links: PMID-39890862
PubMed:
Citation:
show bibtex listing
hide bibtex listing
@article {pmid39890862,
year = {2025},
author = {Nalefski, EA and Sinan, S and Cantera, JL and Kim, AG and Kooistra, RM and Rivera, RE and Janshen, JP and Bhadra, S and Bishop, JD and Ellington, AD and Finklestein, IJ and Madan, D},
title = {Room temperature CRISPR diagnostics for low-resource settings.},
journal = {Scientific reports},
volume = {15},
number = {1},
pages = {3909},
pmid = {39890862},
issn = {2045-2322},
mesh = {Humans ; *CRISPR-Cas Systems ; *Temperature ; Endodeoxyribonucleases/genetics/metabolism ; Human papillomavirus 16/genetics/isolation & purification ; CRISPR-Associated Proteins/genetics ; Bacterial Proteins/genetics ; Female ; Recombinases/metabolism ; Nucleic Acid Amplification Techniques/methods ; Papillomavirus Infections/diagnosis/virology ; Sensitivity and Specificity ; },
abstract = {Maintaining elevated reaction temperatures and multi-step sample preparations increases the costs and complexity of diagnostics, impeding their deployment in low-resource settings. Here, we develop a one-pot, room temperature recombinase polymerase amplification (RPA)-CRISPR reaction that removes these critical challenges. We show that RPA amplification is reduced by several orders of magnitude at 25 °C as compared to 37 °C. Similarly, when coupled to RPA, the performance of multiple Cas12a orthologs, including the widely used LbCas12a, is severely compromised at temperatures below 37 °C. To mitigate these limitations, we identify the ortholog TsCas12a as a highly active nuclease at 25 °C and develop a single-protocol RPA-Cas12a detection reaction with this enzyme. A quantitative kinetic analysis reveals that fast nuclease activation is more critical than higher steady-state trans-cleavage activity for room temperature diagnostic applications. RPA-TsCas12a reactions performed at 25 °C effectively detected HPV-16 in crudely prepared cervical swab samples with high sensitivity and specificity using both optical and lateral flow readouts. The reactions developed herein reduce the complexity and equipment requirements for affordable diagnostics in low- and middle-income countries.},
}
MeSH Terms:
show MeSH Terms
hide MeSH Terms
Humans
*CRISPR-Cas Systems
*Temperature
Endodeoxyribonucleases/genetics/metabolism
Human papillomavirus 16/genetics/isolation & purification
CRISPR-Associated Proteins/genetics
Bacterial Proteins/genetics
Female
Recombinases/metabolism
Nucleic Acid Amplification Techniques/methods
Papillomavirus Infections/diagnosis/virology
Sensitivity and Specificity
RevDate: 2025-02-02
CmpDate: 2025-02-02
Dumbbell probe-bridged CRISPR/Cas13a and nicking-mediated DNA cascade reaction for highly sensitive detection of colorectal cancer-related microRNAs.
Biosensors & bioelectronics, 273:117190.
Colorectal cancer (CRC) is a leading cause of cancer-related deaths globally, necessitating the development of sensitive and minimally invasive diagnostic approaches. In this study, we present a novel diagnostic strategy by integrating dumbbell probe-mediated CRISPR/Cas13a with nicking-induced DNA cascade reaction (DP-bridged Cas13a/NDCR) for highly sensitive microRNA (miRNA) detection. Target miRNA triggers Cas13a-mediated cleavage of the dumbbell probe, releasing an intermediate strand that hybridizes with a methylene blue-labeled hairpin probe on the electrode surface. Nicking enzyme cleaves the formed duplex DNA, triggering a cascade reaction that amplifies the electrochemical signal. Under optimized conditions, the method demonstrates a detection limit of 8.26 fM for miRNA-21, with reliable specificity and long-term stability. Furthermore, integration with machine learning models using multiple miRNA markers improved diagnostic accuracy, differentiating CRC from colorectal polyps and healthy controls with 100% accuracy in clinical validation cohorts. This study highlights the potential of DP-bridged Cas13a/NDCR as a sensitive and accurate diagnostic tool for CRC.
Additional Links: PMID-39862677
Publisher:
PubMed:
Citation:
show bibtex listing
hide bibtex listing
@article {pmid39862677,
year = {2025},
author = {Pei, J and Li, L and Li, C and Li, Z and Wu, Y and Kuang, H and Ma, P and Huang, L and Liu, J and Tian, G},
title = {Dumbbell probe-bridged CRISPR/Cas13a and nicking-mediated DNA cascade reaction for highly sensitive detection of colorectal cancer-related microRNAs.},
journal = {Biosensors & bioelectronics},
volume = {273},
number = {},
pages = {117190},
doi = {10.1016/j.bios.2025.117190},
pmid = {39862677},
issn = {1873-4235},
mesh = {*Colorectal Neoplasms/diagnosis/genetics ; *MicroRNAs/analysis/genetics ; Humans ; *Biosensing Techniques/methods ; *CRISPR-Cas Systems ; Limit of Detection ; DNA/chemistry/genetics ; Electrochemical Techniques/methods ; },
abstract = {Colorectal cancer (CRC) is a leading cause of cancer-related deaths globally, necessitating the development of sensitive and minimally invasive diagnostic approaches. In this study, we present a novel diagnostic strategy by integrating dumbbell probe-mediated CRISPR/Cas13a with nicking-induced DNA cascade reaction (DP-bridged Cas13a/NDCR) for highly sensitive microRNA (miRNA) detection. Target miRNA triggers Cas13a-mediated cleavage of the dumbbell probe, releasing an intermediate strand that hybridizes with a methylene blue-labeled hairpin probe on the electrode surface. Nicking enzyme cleaves the formed duplex DNA, triggering a cascade reaction that amplifies the electrochemical signal. Under optimized conditions, the method demonstrates a detection limit of 8.26 fM for miRNA-21, with reliable specificity and long-term stability. Furthermore, integration with machine learning models using multiple miRNA markers improved diagnostic accuracy, differentiating CRC from colorectal polyps and healthy controls with 100% accuracy in clinical validation cohorts. This study highlights the potential of DP-bridged Cas13a/NDCR as a sensitive and accurate diagnostic tool for CRC.},
}
MeSH Terms:
show MeSH Terms
hide MeSH Terms
*Colorectal Neoplasms/diagnosis/genetics
*MicroRNAs/analysis/genetics
Humans
*Biosensing Techniques/methods
*CRISPR-Cas Systems
Limit of Detection
DNA/chemistry/genetics
Electrochemical Techniques/methods
RevDate: 2025-02-02
CmpDate: 2025-02-02
Non-viral, high throughput genetic engineering of primary immune cells using nanostraw-mediated transfection.
Biomaterials, 317:123079.
Transfection of proteins, mRNA, and chimeric antigen receptor (CAR) transgenes into immune cells remains a critical bottleneck in cell manufacturing. Current methods, such as viruses and bulk electroporation, are hampered by low transfection efficiency, unintended transgene integration, and significant cell perturbation. The Nanostraw Electro-actuated Transfection (NExT) technology offers a solution by using high aspect-ratio nanostraws and localized electric fields to precisely deliver biomolecules into cells with minimal disruption. We demonstrate that NExT can deliver proteins, polysaccharides, and mRNA into primary human CD8[+] and CD4[+] T cells, and achieve CRISPR/Cas9 gene knockout of CXCR4 and TRAC in CD8[+] T cells. We showcase NExT's versatility across a range of primary human immune cells, including CD4[+] T cells, γδ-T cells, dendritic cells, NK cells, Treg cells, macrophages, and neutrophils. Finally, we developed a scalable, high-throughput multiwell NExT system capable of transfecting over 14 million cells and delivering diverse cargoes into multiple cell types from various donors simultaneously. This technology holds promise for streamlining high-throughput screening of allogeneic donors and reducing optimization costs for large-scale CAR-immune cell transfection.
Additional Links: PMID-39842078
Publisher:
PubMed:
Citation:
show bibtex listing
hide bibtex listing
@article {pmid39842078,
year = {2025},
author = {Kumar, ARK and Low, J and Lim, J and Myint, B and Sun, X and Wu, L and Cheng, HS and Yip, S and Ming Cheng, CZ and Manoharan, T and Quek, YJ and Shou, Y and Tian, JS and Ng, YY and Gascoigne, NRJ and Tan, NS and Sugimura, R and Chia, G and Sze Cheung, AM and Yawata, M and Tay, A},
title = {Non-viral, high throughput genetic engineering of primary immune cells using nanostraw-mediated transfection.},
journal = {Biomaterials},
volume = {317},
number = {},
pages = {123079},
doi = {10.1016/j.biomaterials.2024.123079},
pmid = {39842078},
issn = {1878-5905},
mesh = {Humans ; *Transfection/methods ; *CRISPR-Cas Systems/genetics ; *Genetic Engineering/methods ; CD8-Positive T-Lymphocytes/immunology ; Electroporation/methods ; CD4-Positive T-Lymphocytes/immunology ; },
abstract = {Transfection of proteins, mRNA, and chimeric antigen receptor (CAR) transgenes into immune cells remains a critical bottleneck in cell manufacturing. Current methods, such as viruses and bulk electroporation, are hampered by low transfection efficiency, unintended transgene integration, and significant cell perturbation. The Nanostraw Electro-actuated Transfection (NExT) technology offers a solution by using high aspect-ratio nanostraws and localized electric fields to precisely deliver biomolecules into cells with minimal disruption. We demonstrate that NExT can deliver proteins, polysaccharides, and mRNA into primary human CD8[+] and CD4[+] T cells, and achieve CRISPR/Cas9 gene knockout of CXCR4 and TRAC in CD8[+] T cells. We showcase NExT's versatility across a range of primary human immune cells, including CD4[+] T cells, γδ-T cells, dendritic cells, NK cells, Treg cells, macrophages, and neutrophils. Finally, we developed a scalable, high-throughput multiwell NExT system capable of transfecting over 14 million cells and delivering diverse cargoes into multiple cell types from various donors simultaneously. This technology holds promise for streamlining high-throughput screening of allogeneic donors and reducing optimization costs for large-scale CAR-immune cell transfection.},
}
MeSH Terms:
show MeSH Terms
hide MeSH Terms
Humans
*Transfection/methods
*CRISPR-Cas Systems/genetics
*Genetic Engineering/methods
CD8-Positive T-Lymphocytes/immunology
Electroporation/methods
CD4-Positive T-Lymphocytes/immunology
RevDate: 2025-02-02
CmpDate: 2025-02-02
A conformational switch-controlled RNA sensor based on orthogonal dCas12a for RNA imaging in live cells.
Biosensors & bioelectronics, 273:117185.
RNA imaging technology is essential for understanding the complex RNA regulatory mechanisms and serves as a powerful tool for disease diagnosis. However, conventional RNA imaging methods often require multiple fluorescent tags for the specific labeling of individual targets, complicating both the imaging process and subsequent analysis. Herein, we develop an RNA sensor that integrates a blocked CRISPR RNA (crRNA)-based conformational switch with a controllable CRISPR activation (CRISPRa) system and apply for RNA imaging. By leveraging nuclease-inactive Cas12a (dCas12a)-mediated processing of precursor crRNA (pre-crRNA) and the orthogonality of dCas12a from different bacteria, our sensor establishes an artificial link between two unrelated RNA targets, enabling cells to sense one RNA target and image another with a single fluorescent signal. By visualizing a single target for dual-target analysis, our method significantly reduces the reliance on multiple fluorescent tags. Our sensor provides a new platform for RNA imaging, enhancing both biomedical research and the development of advanced molecular diagnostics.
Additional Links: PMID-39842058
Publisher:
PubMed:
Citation:
show bibtex listing
hide bibtex listing
@article {pmid39842058,
year = {2025},
author = {Jia, HY and Yao, SY and Li, YF and Ye, BC and Yin, BC},
title = {A conformational switch-controlled RNA sensor based on orthogonal dCas12a for RNA imaging in live cells.},
journal = {Biosensors & bioelectronics},
volume = {273},
number = {},
pages = {117185},
doi = {10.1016/j.bios.2025.117185},
pmid = {39842058},
issn = {1873-4235},
mesh = {*Biosensing Techniques/methods ; Humans ; *RNA/chemistry/genetics ; *CRISPR-Cas Systems ; CRISPR-Associated Proteins/chemistry ; Bacterial Proteins/chemistry/genetics ; Nucleic Acid Conformation ; Endodeoxyribonucleases/chemistry ; Fluorescent Dyes/chemistry ; },
abstract = {RNA imaging technology is essential for understanding the complex RNA regulatory mechanisms and serves as a powerful tool for disease diagnosis. However, conventional RNA imaging methods often require multiple fluorescent tags for the specific labeling of individual targets, complicating both the imaging process and subsequent analysis. Herein, we develop an RNA sensor that integrates a blocked CRISPR RNA (crRNA)-based conformational switch with a controllable CRISPR activation (CRISPRa) system and apply for RNA imaging. By leveraging nuclease-inactive Cas12a (dCas12a)-mediated processing of precursor crRNA (pre-crRNA) and the orthogonality of dCas12a from different bacteria, our sensor establishes an artificial link between two unrelated RNA targets, enabling cells to sense one RNA target and image another with a single fluorescent signal. By visualizing a single target for dual-target analysis, our method significantly reduces the reliance on multiple fluorescent tags. Our sensor provides a new platform for RNA imaging, enhancing both biomedical research and the development of advanced molecular diagnostics.},
}
MeSH Terms:
show MeSH Terms
hide MeSH Terms
*Biosensing Techniques/methods
Humans
*RNA/chemistry/genetics
*CRISPR-Cas Systems
CRISPR-Associated Proteins/chemistry
Bacterial Proteins/chemistry/genetics
Nucleic Acid Conformation
Endodeoxyribonucleases/chemistry
Fluorescent Dyes/chemistry
RevDate: 2025-02-02
CmpDate: 2025-02-02
Integrating commercial personal glucose meter with peroxidase-mimic DNAzyme to develop a versatile point-of-care biosensing platform.
Biosensors & bioelectronics, 273:117171.
The development of point-of-care testing (POCT) methods is highly desirable in molecular detection, as they enable disease diagnosis and biomarker monitoring on-site or at home. Repurposing existing POCT devices to detect diverse biomarkers is an economical way to develop new devices for POCT use. Personal glucose meter (PGM) is one of the most used off-the-shelf POCT devices that has been reused to detect non-glucose targets. However, developing a label-free, user-friendly, and cost-effective general PGM-based sensing platform remains a great challenge, primarily due to the reliance on protein enzymes in most existing signal transducing strategies. To overcome the challenges, we herein developed a DNAzyme-based signal transduction strategy that bridges non-glucose signals to PGM readouts. By integrating this strategy with CRISPR/Cas12a-mediated target sensing, we successfully established a simple and versatile platform (CaG-PGM) for biosensing. The utility of CaG-PGM in the detection of nucleic acid targets was successfully validated by detecting Monkeypox virus DNA and SARS-CoV-2 RNA with high sensitivity and specificity. We further demonstrated its generality in detecting non-nucleic acid targets including protein and small molecule. In conclusion, this study provides a cheap and effective strategy for repurposing PGM as a general biosensing platform and sheds new light on translating functional nucleic acids for POCT applications.
Additional Links: PMID-39837235
Publisher:
PubMed:
Citation:
show bibtex listing
hide bibtex listing
@article {pmid39837235,
year = {2025},
author = {Ding, S and Dong, J and Shi, J and Ren, K and Cui, X and Shi, Z and Li, N and Xiang, Y and Du, F and Tang, Z},
title = {Integrating commercial personal glucose meter with peroxidase-mimic DNAzyme to develop a versatile point-of-care biosensing platform.},
journal = {Biosensors & bioelectronics},
volume = {273},
number = {},
pages = {117171},
doi = {10.1016/j.bios.2025.117171},
pmid = {39837235},
issn = {1873-4235},
mesh = {*DNA, Catalytic/chemistry ; *Biosensing Techniques/instrumentation/methods ; Humans ; *SARS-CoV-2/isolation & purification ; *COVID-19/diagnosis/virology ; *Blood Glucose Self-Monitoring/instrumentation ; Point-of-Care Testing ; Point-of-Care Systems ; Peroxidase/chemistry ; Glucose/analysis ; Blood Glucose/analysis ; CRISPR-Cas Systems ; RNA, Viral/analysis ; Limit of Detection ; },
abstract = {The development of point-of-care testing (POCT) methods is highly desirable in molecular detection, as they enable disease diagnosis and biomarker monitoring on-site or at home. Repurposing existing POCT devices to detect diverse biomarkers is an economical way to develop new devices for POCT use. Personal glucose meter (PGM) is one of the most used off-the-shelf POCT devices that has been reused to detect non-glucose targets. However, developing a label-free, user-friendly, and cost-effective general PGM-based sensing platform remains a great challenge, primarily due to the reliance on protein enzymes in most existing signal transducing strategies. To overcome the challenges, we herein developed a DNAzyme-based signal transduction strategy that bridges non-glucose signals to PGM readouts. By integrating this strategy with CRISPR/Cas12a-mediated target sensing, we successfully established a simple and versatile platform (CaG-PGM) for biosensing. The utility of CaG-PGM in the detection of nucleic acid targets was successfully validated by detecting Monkeypox virus DNA and SARS-CoV-2 RNA with high sensitivity and specificity. We further demonstrated its generality in detecting non-nucleic acid targets including protein and small molecule. In conclusion, this study provides a cheap and effective strategy for repurposing PGM as a general biosensing platform and sheds new light on translating functional nucleic acids for POCT applications.},
}
MeSH Terms:
show MeSH Terms
hide MeSH Terms
*DNA, Catalytic/chemistry
*Biosensing Techniques/instrumentation/methods
Humans
*SARS-CoV-2/isolation & purification
*COVID-19/diagnosis/virology
*Blood Glucose Self-Monitoring/instrumentation
Point-of-Care Testing
Point-of-Care Systems
Peroxidase/chemistry
Glucose/analysis
Blood Glucose/analysis
CRISPR-Cas Systems
RNA, Viral/analysis
Limit of Detection
RevDate: 2025-02-01
CmpDate: 2025-02-01
Unlocking the potential of CRISPR-Cas9 for cystic fibrosis: A systematic literature review.
Gene, 942:149257.
CRISPR-Cas9 technology has revolutionized genetic engineering, offering precise and efficient genome editing capabilities. This review explores the application of CRISPR-Cas9 for cystic fibrosis (CF), particularly targeting mutations in the CFTR gene. CF is a multiorgan disease primarily affecting the lungs, gastrointestinal system (e.g., CF-related diabetes (CFRD), CF-associated liver disease (CFLD)), bones (CF-bone disease), and the reproductive system. CF, a genetic disorder characterized by defective ion transport leading to thick mucus accumulation, is often caused by mutations like ΔF508 in the CFTR gene. This review employs a systematic methodology, incorporating an extensive literature search across multiple academic databases, including PubMed, Web of Science, and ScienceDirect, to identify 40 high-quality studies focused on CRISPR-Cas9 applications for CFTR gene editing. The data collection process involved predefined inclusion criteria targeting experimental approaches, gene-editing outcomes, delivery methods, and verification techniques. Data analysis synthesized findings on editing efficiency, off-target effects, and delivery system optimization to present a comprehensive overview of the field. The review highlights the historical development of CRISPR-Cas9, its mechanism, and its transformative role in genetic engineering and medicine. A detailed examination of CRISPR-Cas9's application in CFTR gene correction emphasizes the potential for therapeutic interventions while addressing challenges such as off-target effects, delivery efficiency, and ethical considerations. Future directions include optimizing delivery systems, integrating advanced editing tools like prime and base editing, and expanding personalized medicine approaches to improve treatment outcomes. By systematically analyzing the current landscape, this review provides a foundation for advancing CRISPR-Cas9 technologies for cystic fibrosis treatment and related disorders.
Additional Links: PMID-39832688
Publisher:
PubMed:
Citation:
show bibtex listing
hide bibtex listing
@article {pmid39832688,
year = {2025},
author = {Harris, H and Kittur, J},
title = {Unlocking the potential of CRISPR-Cas9 for cystic fibrosis: A systematic literature review.},
journal = {Gene},
volume = {942},
number = {},
pages = {149257},
doi = {10.1016/j.gene.2025.149257},
pmid = {39832688},
issn = {1879-0038},
mesh = {*Cystic Fibrosis/genetics/therapy ; Humans ; *CRISPR-Cas Systems ; *Gene Editing/methods ; *Cystic Fibrosis Transmembrane Conductance Regulator/genetics ; *Genetic Therapy/methods ; Animals ; Mutation ; },
abstract = {CRISPR-Cas9 technology has revolutionized genetic engineering, offering precise and efficient genome editing capabilities. This review explores the application of CRISPR-Cas9 for cystic fibrosis (CF), particularly targeting mutations in the CFTR gene. CF is a multiorgan disease primarily affecting the lungs, gastrointestinal system (e.g., CF-related diabetes (CFRD), CF-associated liver disease (CFLD)), bones (CF-bone disease), and the reproductive system. CF, a genetic disorder characterized by defective ion transport leading to thick mucus accumulation, is often caused by mutations like ΔF508 in the CFTR gene. This review employs a systematic methodology, incorporating an extensive literature search across multiple academic databases, including PubMed, Web of Science, and ScienceDirect, to identify 40 high-quality studies focused on CRISPR-Cas9 applications for CFTR gene editing. The data collection process involved predefined inclusion criteria targeting experimental approaches, gene-editing outcomes, delivery methods, and verification techniques. Data analysis synthesized findings on editing efficiency, off-target effects, and delivery system optimization to present a comprehensive overview of the field. The review highlights the historical development of CRISPR-Cas9, its mechanism, and its transformative role in genetic engineering and medicine. A detailed examination of CRISPR-Cas9's application in CFTR gene correction emphasizes the potential for therapeutic interventions while addressing challenges such as off-target effects, delivery efficiency, and ethical considerations. Future directions include optimizing delivery systems, integrating advanced editing tools like prime and base editing, and expanding personalized medicine approaches to improve treatment outcomes. By systematically analyzing the current landscape, this review provides a foundation for advancing CRISPR-Cas9 technologies for cystic fibrosis treatment and related disorders.},
}
MeSH Terms:
show MeSH Terms
hide MeSH Terms
*Cystic Fibrosis/genetics/therapy
Humans
*CRISPR-Cas Systems
*Gene Editing/methods
*Cystic Fibrosis Transmembrane Conductance Regulator/genetics
*Genetic Therapy/methods
Animals
Mutation
RevDate: 2025-02-02
CmpDate: 2025-02-02
CIMNE-CRISPR: A novel amplification-free diagnostic for rapid early detection of African Swine Fever Virus.
Biosensors & bioelectronics, 273:117154.
African Swine Fever Virus (ASFV) is a highly contagious pathogen with nearly 100% mortality in swine, causing severe global economic loss. Current detection methods rely on nucleic acid amplification, which requires specialized equipment and skilled operators, limiting accessibility in resource-constrained settings. To address these challenges, we developed the Covalently Immobilized Magnetic Nanoparticles Enhanced CRISPR (CIMNE-CRISPR) system. This amplification-free diagnostic system seamlessly combines target recognition, sequence-specific enrichment, and signal generation. This approach uses covalent immobilization of CRISPR-LbCas12a-crRNA complexes on Fe3O4@SiO2 core-shell magnetic nanoparticles, which improves enzyme specificity and robustness over traditional adsorption. The CIMNE-CRISPR assay reached a limit of detection (LOD) of 8.1 × 10[4] copies/μL and a limit of quantification (LOQ) of 4.2 × 10[5] copies/μL, with a dynamic range spanning 10[5] to 10[10] copies/μL and a matrix factor of 100.29% in porcine plasma. It maintained great specificity and accurately detecting 10[5] copies/μL of ASFV DNA even with high mutant concentrations (10[13] copies/μL). The method demonstrated decent reproducibility across different nanoparticle synthesis batches, with an RSD of 9.63% and recovery rates between 97% and 103%, and features rapid processing well-suited for field diagnostics. Overall, this system's cost-effectiveness, simplicity, and reliability highlight its potential to pave the way for advanced CRISPR-based diagnostics, particularly for diverse viral and bacterial targets in agricultural, environmental, and zoonotic disease contexts.
Additional Links: PMID-39826273
Publisher:
PubMed:
Citation:
show bibtex listing
hide bibtex listing
@article {pmid39826273,
year = {2025},
author = {Pal, T and Liu, Z and Chen, J},
title = {CIMNE-CRISPR: A novel amplification-free diagnostic for rapid early detection of African Swine Fever Virus.},
journal = {Biosensors & bioelectronics},
volume = {273},
number = {},
pages = {117154},
doi = {10.1016/j.bios.2025.117154},
pmid = {39826273},
issn = {1873-4235},
support = {R35 GM147069/GM/NIGMS NIH HHS/United States ; },
mesh = {*African Swine Fever Virus/isolation & purification/genetics ; Animals ; Swine ; *African Swine Fever/diagnosis/virology/blood ; *Biosensing Techniques/methods ; *Limit of Detection ; CRISPR-Cas Systems ; Magnetite Nanoparticles/chemistry ; DNA, Viral/analysis/genetics ; Reproducibility of Results ; },
abstract = {African Swine Fever Virus (ASFV) is a highly contagious pathogen with nearly 100% mortality in swine, causing severe global economic loss. Current detection methods rely on nucleic acid amplification, which requires specialized equipment and skilled operators, limiting accessibility in resource-constrained settings. To address these challenges, we developed the Covalently Immobilized Magnetic Nanoparticles Enhanced CRISPR (CIMNE-CRISPR) system. This amplification-free diagnostic system seamlessly combines target recognition, sequence-specific enrichment, and signal generation. This approach uses covalent immobilization of CRISPR-LbCas12a-crRNA complexes on Fe3O4@SiO2 core-shell magnetic nanoparticles, which improves enzyme specificity and robustness over traditional adsorption. The CIMNE-CRISPR assay reached a limit of detection (LOD) of 8.1 × 10[4] copies/μL and a limit of quantification (LOQ) of 4.2 × 10[5] copies/μL, with a dynamic range spanning 10[5] to 10[10] copies/μL and a matrix factor of 100.29% in porcine plasma. It maintained great specificity and accurately detecting 10[5] copies/μL of ASFV DNA even with high mutant concentrations (10[13] copies/μL). The method demonstrated decent reproducibility across different nanoparticle synthesis batches, with an RSD of 9.63% and recovery rates between 97% and 103%, and features rapid processing well-suited for field diagnostics. Overall, this system's cost-effectiveness, simplicity, and reliability highlight its potential to pave the way for advanced CRISPR-based diagnostics, particularly for diverse viral and bacterial targets in agricultural, environmental, and zoonotic disease contexts.},
}
MeSH Terms:
show MeSH Terms
hide MeSH Terms
*African Swine Fever Virus/isolation & purification/genetics
Animals
Swine
*African Swine Fever/diagnosis/virology/blood
*Biosensing Techniques/methods
*Limit of Detection
CRISPR-Cas Systems
Magnetite Nanoparticles/chemistry
DNA, Viral/analysis/genetics
Reproducibility of Results
RevDate: 2025-02-02
CmpDate: 2025-02-02
The dCas9/crRNA linked immunological assay (dCLISA) for sensitive, accurate, and facile drug resistance gene analysis.
Biosensors & bioelectronics, 273:117147.
The rapid and reliable diagnosis of methicillin-resistant Staphylococcus aureus (MRSA) is essential for preventing the spread of MRSA infections and guiding therapeutic strategies. However, there is still a huge challenge in further simplifying MRSA detection procedures and improving detection selectivity to reduce false-positive results. In this study, we developed a derivative CRISPR-associated protein 9/CRISPR-derived RNA Linked Immunological Assay (dCLISA) for the sensitive and specific detection of MRSA. This technique utilizes two dCas9/crRNA complexes as specific targeting agents and employs a color reaction mediated by a hybridization chain reaction for signal output. The dCLISA method offers certain benefits compared to monoclonal antibodies in traditional immunoassays, primarily due to its capacity to selectively interact with target gene and its high sensitivity from the hybridization chain reaction process. Therefore, the minimum detectable concentration of dCLISA was 8.5 cfu/mL. Unlike traditional gene analysis approaches, target gene sequences in cell lysates can be directly detected by dCLISA within 60 min with high sensitivity without genomic material extraction. In addition, the absorbance intensity of the MRSA cell lysate was significantly higher than that of methicillin-susceptible S. aureus (MSSA) indicates the clinical application potential. This study demonstrates that the dCLISA is a simple, rapid, sensitive, and specific method, which can be directly used at the point of care to analyze drug resistance in bacteria, including MRSA. Moreover, dCLISA can be utilized for other bacteria detection by merely modifying the crRNA sequence.
Additional Links: PMID-39823857
Publisher:
PubMed:
Citation:
show bibtex listing
hide bibtex listing
@article {pmid39823857,
year = {2025},
author = {Fan, X and Gao, Z and Ling, D and Wang, D and Cui, Y and Du, H and Zhou, X},
title = {The dCas9/crRNA linked immunological assay (dCLISA) for sensitive, accurate, and facile drug resistance gene analysis.},
journal = {Biosensors & bioelectronics},
volume = {273},
number = {},
pages = {117147},
doi = {10.1016/j.bios.2025.117147},
pmid = {39823857},
issn = {1873-4235},
mesh = {*Methicillin-Resistant Staphylococcus aureus/isolation & purification/genetics ; Humans ; *Biosensing Techniques/methods ; *Staphylococcal Infections/microbiology/immunology ; *CRISPR-Cas Systems ; CRISPR-Associated Protein 9/genetics ; Immunoassay/methods ; Drug Resistance, Bacterial/genetics ; },
abstract = {The rapid and reliable diagnosis of methicillin-resistant Staphylococcus aureus (MRSA) is essential for preventing the spread of MRSA infections and guiding therapeutic strategies. However, there is still a huge challenge in further simplifying MRSA detection procedures and improving detection selectivity to reduce false-positive results. In this study, we developed a derivative CRISPR-associated protein 9/CRISPR-derived RNA Linked Immunological Assay (dCLISA) for the sensitive and specific detection of MRSA. This technique utilizes two dCas9/crRNA complexes as specific targeting agents and employs a color reaction mediated by a hybridization chain reaction for signal output. The dCLISA method offers certain benefits compared to monoclonal antibodies in traditional immunoassays, primarily due to its capacity to selectively interact with target gene and its high sensitivity from the hybridization chain reaction process. Therefore, the minimum detectable concentration of dCLISA was 8.5 cfu/mL. Unlike traditional gene analysis approaches, target gene sequences in cell lysates can be directly detected by dCLISA within 60 min with high sensitivity without genomic material extraction. In addition, the absorbance intensity of the MRSA cell lysate was significantly higher than that of methicillin-susceptible S. aureus (MSSA) indicates the clinical application potential. This study demonstrates that the dCLISA is a simple, rapid, sensitive, and specific method, which can be directly used at the point of care to analyze drug resistance in bacteria, including MRSA. Moreover, dCLISA can be utilized for other bacteria detection by merely modifying the crRNA sequence.},
}
MeSH Terms:
show MeSH Terms
hide MeSH Terms
*Methicillin-Resistant Staphylococcus aureus/isolation & purification/genetics
Humans
*Biosensing Techniques/methods
*Staphylococcal Infections/microbiology/immunology
*CRISPR-Cas Systems
CRISPR-Associated Protein 9/genetics
Immunoassay/methods
Drug Resistance, Bacterial/genetics
RevDate: 2025-02-02
CmpDate: 2025-02-02
Nano-polymeric platinum activates PAR2 gene editing to suppress tumor metastasis.
Biomaterials, 317:123090.
Metastasis as the hallmark of cancer preferentially contributes to tumor recurrence and therapy resistance, aggrandizing the lethality of patients with cancer. Despite their robust suppressions of tumor progression, chemotherapeutics failed to attenuate cancer cell migration and even triggered pro-metastatic effects. In parallel, protease-activated receptor 2 (PAR2), a member of the G protein-coupled receptor subfamily, actively participates in cancer metastasis via multiple signal transduction pathways. CRISPR/Cas9 that is a dominating genome editing tool can evoke PAR2 knockout to inhibit cancer metastasis. However, the absence of valid delivery systems largely limits its efficacy. Herein, we nanosized polymeric platinum (NanoPt) as therapeutical drug carries to deliver CRISPR/Cas9 to elicit genome editing of PAR2, which drastically augmented anti-metastatic effects and alleviated systematic toxicity of platinum-based treatment in vitro and in vivo. More importantly, the NanoPt@Cas9-PAR2 initiated PAR2 deficiency to mechanistically attenuate EMT process and ferroptosis via RAGE/ERK signalling, consequently preventing cancer cell migration. Our findings indicate that NanoPt@Cas9-PAR2 that mitigated PAR2 signalling and cytotoxic effects of platinum could be a safe and powerful all-in-one combinatorial strategy for cancer treatment.
Additional Links: PMID-39799696
Publisher:
PubMed:
Citation:
show bibtex listing
hide bibtex listing
@article {pmid39799696,
year = {2025},
author = {Jiang, Y and Li, Y and Zheng, D and Du, X and Yang, H and Wang, C and Zhao, M and Xiao, H and Zhang, L and Li, X and Shi, S},
title = {Nano-polymeric platinum activates PAR2 gene editing to suppress tumor metastasis.},
journal = {Biomaterials},
volume = {317},
number = {},
pages = {123090},
doi = {10.1016/j.biomaterials.2025.123090},
pmid = {39799696},
issn = {1878-5905},
mesh = {*Receptor, PAR-2/metabolism/genetics ; Humans ; Animals ; *Platinum/chemistry/pharmacology/therapeutic use ; *Neoplasm Metastasis ; *Gene Editing ; Cell Line, Tumor ; Mice ; Cell Movement/drug effects ; CRISPR-Cas Systems ; Polymers/chemistry ; Mice, Nude ; Mice, Inbred BALB C ; Antineoplastic Agents/pharmacology/therapeutic use ; Female ; Nanoparticles/chemistry ; },
abstract = {Metastasis as the hallmark of cancer preferentially contributes to tumor recurrence and therapy resistance, aggrandizing the lethality of patients with cancer. Despite their robust suppressions of tumor progression, chemotherapeutics failed to attenuate cancer cell migration and even triggered pro-metastatic effects. In parallel, protease-activated receptor 2 (PAR2), a member of the G protein-coupled receptor subfamily, actively participates in cancer metastasis via multiple signal transduction pathways. CRISPR/Cas9 that is a dominating genome editing tool can evoke PAR2 knockout to inhibit cancer metastasis. However, the absence of valid delivery systems largely limits its efficacy. Herein, we nanosized polymeric platinum (NanoPt) as therapeutical drug carries to deliver CRISPR/Cas9 to elicit genome editing of PAR2, which drastically augmented anti-metastatic effects and alleviated systematic toxicity of platinum-based treatment in vitro and in vivo. More importantly, the NanoPt@Cas9-PAR2 initiated PAR2 deficiency to mechanistically attenuate EMT process and ferroptosis via RAGE/ERK signalling, consequently preventing cancer cell migration. Our findings indicate that NanoPt@Cas9-PAR2 that mitigated PAR2 signalling and cytotoxic effects of platinum could be a safe and powerful all-in-one combinatorial strategy for cancer treatment.},
}
MeSH Terms:
show MeSH Terms
hide MeSH Terms
*Receptor, PAR-2/metabolism/genetics
Humans
Animals
*Platinum/chemistry/pharmacology/therapeutic use
*Neoplasm Metastasis
*Gene Editing
Cell Line, Tumor
Mice
Cell Movement/drug effects
CRISPR-Cas Systems
Polymers/chemistry
Mice, Nude
Mice, Inbred BALB C
Antineoplastic Agents/pharmacology/therapeutic use
Female
Nanoparticles/chemistry
RevDate: 2025-02-01
CmpDate: 2025-02-01
Enhancing the production of L-proline in recombinant Escherichia coli BL21 by metabolic engineering.
Preparative biochemistry & biotechnology, 55(2):187-195.
L-proline is widely used in the fields of food, medicine and agriculture, and is also an important raw material for the synthesis of trans-4-hydroxy-L-proline. In this study, enhancing the production of L-proline by metabolic engineering was investigated. Three genes, proB, proA and proC, were introduced into Escherichia coli BL21 by molecular biology technology to increase the metabolic flow of L-proline from glucose. The genes putP and proP related to the proline transfer were knocked out by CRISPR/Cas9 gene editing technology to weaken the feedback inhibition of proB to increase the production of L-proline. The fermentation curves of the engineered strain at different glucose concentrations were determined, and a glucose concentration of 10 g/L was chosen to expand the batch culture to 1 L shake flask. Ultimately, through these efforts, the titer of L-proline reached 832.19 mg/L in intermittent glucose addition fermentation in a 1 L shake flask.
Additional Links: PMID-38984870
Publisher:
PubMed:
Citation:
show bibtex listing
hide bibtex listing
@article {pmid38984870,
year = {2025},
author = {Lu, J and Fu, B and Zhu, Z and Yan, C and Guan, F and Wang, P and Yu, P},
title = {Enhancing the production of L-proline in recombinant Escherichia coli BL21 by metabolic engineering.},
journal = {Preparative biochemistry & biotechnology},
volume = {55},
number = {2},
pages = {187-195},
doi = {10.1080/10826068.2024.2378104},
pmid = {38984870},
issn = {1532-2297},
mesh = {*Proline/metabolism ; *Escherichia coli/genetics/metabolism ; *Metabolic Engineering/methods ; *Glucose/metabolism ; *CRISPR-Cas Systems ; *Fermentation ; Gene Editing/methods ; Escherichia coli Proteins/genetics/metabolism ; Batch Cell Culture Techniques ; },
abstract = {L-proline is widely used in the fields of food, medicine and agriculture, and is also an important raw material for the synthesis of trans-4-hydroxy-L-proline. In this study, enhancing the production of L-proline by metabolic engineering was investigated. Three genes, proB, proA and proC, were introduced into Escherichia coli BL21 by molecular biology technology to increase the metabolic flow of L-proline from glucose. The genes putP and proP related to the proline transfer were knocked out by CRISPR/Cas9 gene editing technology to weaken the feedback inhibition of proB to increase the production of L-proline. The fermentation curves of the engineered strain at different glucose concentrations were determined, and a glucose concentration of 10 g/L was chosen to expand the batch culture to 1 L shake flask. Ultimately, through these efforts, the titer of L-proline reached 832.19 mg/L in intermittent glucose addition fermentation in a 1 L shake flask.},
}
MeSH Terms:
show MeSH Terms
hide MeSH Terms
*Proline/metabolism
*Escherichia coli/genetics/metabolism
*Metabolic Engineering/methods
*Glucose/metabolism
*CRISPR-Cas Systems
*Fermentation
Gene Editing/methods
Escherichia coli Proteins/genetics/metabolism
Batch Cell Culture Techniques
RevDate: 2025-01-31
From bench to bedside: Developing CRISPR/Cas-based therapy for ocular diseases.
Pharmacological research pii:S1043-6618(25)00063-5 [Epub ahead of print].
Vision-threatening disorders, including both hereditary and multifactorial ocular diseases, necessitate innovative therapeutic approaches. The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated protein (Cas) has emerged as a promising tool for treating ocular diseases through gene editing and expression regulation. This system has contributed to the development of representative disease models, including animal models, organoids, and cell lines, thereby facilitating investigations into the pathogenesis of disease-related genes. Besides, therapeutic applications of CRISPR/Cas have been extensively explored in preclinical in vitro and in vivo studies, targeting various ocular conditions, such as retinitis pigmentosa, Leber congenital amaurosis, Usher syndrome, fundus neovascular diseases, glaucoma, and corneal diseases. Recent advancements have demonstrated the technology's potential to restore cellular homeostasis and alleviate disease phenotypes, thereby prompting a variety of clinical trials. To date, active trials include treatments for primary open angle glaucoma with MYOC mutations, refractory herpetic viral keratitis, CEP290-associated inherited retinal degenerations, neovascular age-related macular degeneration, and retinitis pigmentosa with RHO mutations. However, challenges remain, primarily concerning off-target effects, immunogenicity, ethical considerations, and regulatory particularity. To reach higher safety and efficiency before truly transitioning from bench to bedside, future research should concentrate on improving the specificity and efficacy of Cas proteins, optimizing delivery vectors, and broadening the applicability of therapeutic targets. This review summarizes the application strategies and delivery methods of CRISPR/Cas, discusses recent progress in CRISPR/Cas-based disease models and therapies, and provides an overview of the landscape of clinical trials. Current obstacles and future directions regarding the bench-to-bedside transition are also discussed.
Additional Links: PMID-39889868
Publisher:
PubMed:
Citation:
show bibtex listing
hide bibtex listing
@article {pmid39889868,
year = {2025},
author = {Zhao, Q and Wei, L and Chen, Y},
title = {From bench to bedside: Developing CRISPR/Cas-based therapy for ocular diseases.},
journal = {Pharmacological research},
volume = {},
number = {},
pages = {107638},
doi = {10.1016/j.phrs.2025.107638},
pmid = {39889868},
issn = {1096-1186},
abstract = {Vision-threatening disorders, including both hereditary and multifactorial ocular diseases, necessitate innovative therapeutic approaches. The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated protein (Cas) has emerged as a promising tool for treating ocular diseases through gene editing and expression regulation. This system has contributed to the development of representative disease models, including animal models, organoids, and cell lines, thereby facilitating investigations into the pathogenesis of disease-related genes. Besides, therapeutic applications of CRISPR/Cas have been extensively explored in preclinical in vitro and in vivo studies, targeting various ocular conditions, such as retinitis pigmentosa, Leber congenital amaurosis, Usher syndrome, fundus neovascular diseases, glaucoma, and corneal diseases. Recent advancements have demonstrated the technology's potential to restore cellular homeostasis and alleviate disease phenotypes, thereby prompting a variety of clinical trials. To date, active trials include treatments for primary open angle glaucoma with MYOC mutations, refractory herpetic viral keratitis, CEP290-associated inherited retinal degenerations, neovascular age-related macular degeneration, and retinitis pigmentosa with RHO mutations. However, challenges remain, primarily concerning off-target effects, immunogenicity, ethical considerations, and regulatory particularity. To reach higher safety and efficiency before truly transitioning from bench to bedside, future research should concentrate on improving the specificity and efficacy of Cas proteins, optimizing delivery vectors, and broadening the applicability of therapeutic targets. This review summarizes the application strategies and delivery methods of CRISPR/Cas, discusses recent progress in CRISPR/Cas-based disease models and therapies, and provides an overview of the landscape of clinical trials. Current obstacles and future directions regarding the bench-to-bedside transition are also discussed.},
}
RevDate: 2025-01-31
Precision engineering of the probiotic Escherichia coli Nissle 1917 with prime editing.
Applied and environmental microbiology [Epub ahead of print].
CRISPR-Cas systems are transforming precision medicine with engineered probiotics as next-generation diagnostics and therapeutics. To promote human health and treat disease, engineering probiotic bacteria demands maximal versatility to enable non-natural functionalities while minimizing undesired genomic interferences. Here, we present a streamlined prime editing approach tailored for probiotic Escherichia coli Nissle 1917 utilizing only essential genetic modules, including Cas9 nickase from Streptococcus pyogenes, a codon-optimized reverse transcriptase, and a prime editing guide RNA, and an optimized workflow with longer induction. As a result, we achieved all types of prime editing in every individual round of experiments with efficiencies of 25.0%, 52.0%, and 66.7% for DNA deletion, insertion, and substitution, respectively. A comprehensive evaluation of off-target effects revealed a significant reduction in unintended mutations, particularly in comparison to two different base editing methods. Leveraging the prime editing system, we inserted a unique DNA sequence to barcode the edited strain and established an antibiotic-resistance-gene-free platform to enable non-natural functionalities. Our prime editing strategy presents a CRISPR-Cas system that can be readily implemented in any laboratories with the basic CRISPR setups, paving the way for future innovations in engineered probiotics.IMPORTANCEOne ultimate goal of gene editing is to introduce designed DNA variations at specific loci in living organisms with minimal unintended interferences in the genome. Achieving this goal is especially critical for creating engineered probiotics as living diagnostics and therapeutics to promote human health and treat diseases. In this endeavor, we report a customized prime editing system for precision engineering of probiotic Escherichia coli Nissle 1917. With such a system, we developed a barcoding system for tracking engineered strains, and we built an antibiotic-resistance-gene-free platform to enable non-natural functionalities. We provide not only a powerful gene editing approach for probiotic bacteria but also new insights into the advancement of innovative CRISPR-Cas systems.
Additional Links: PMID-39887239
Publisher:
PubMed:
Citation:
show bibtex listing
hide bibtex listing
@article {pmid39887239,
year = {2025},
author = {Chen, P-R and Wei, Y and Li, X and Yu, H-Y and Wang, S-G and Yuan, X-Z and Xia, P-F},
title = {Precision engineering of the probiotic Escherichia coli Nissle 1917 with prime editing.},
journal = {Applied and environmental microbiology},
volume = {},
number = {},
pages = {e0003125},
doi = {10.1128/aem.00031-25},
pmid = {39887239},
issn = {1098-5336},
abstract = {CRISPR-Cas systems are transforming precision medicine with engineered probiotics as next-generation diagnostics and therapeutics. To promote human health and treat disease, engineering probiotic bacteria demands maximal versatility to enable non-natural functionalities while minimizing undesired genomic interferences. Here, we present a streamlined prime editing approach tailored for probiotic Escherichia coli Nissle 1917 utilizing only essential genetic modules, including Cas9 nickase from Streptococcus pyogenes, a codon-optimized reverse transcriptase, and a prime editing guide RNA, and an optimized workflow with longer induction. As a result, we achieved all types of prime editing in every individual round of experiments with efficiencies of 25.0%, 52.0%, and 66.7% for DNA deletion, insertion, and substitution, respectively. A comprehensive evaluation of off-target effects revealed a significant reduction in unintended mutations, particularly in comparison to two different base editing methods. Leveraging the prime editing system, we inserted a unique DNA sequence to barcode the edited strain and established an antibiotic-resistance-gene-free platform to enable non-natural functionalities. Our prime editing strategy presents a CRISPR-Cas system that can be readily implemented in any laboratories with the basic CRISPR setups, paving the way for future innovations in engineered probiotics.IMPORTANCEOne ultimate goal of gene editing is to introduce designed DNA variations at specific loci in living organisms with minimal unintended interferences in the genome. Achieving this goal is especially critical for creating engineered probiotics as living diagnostics and therapeutics to promote human health and treat diseases. In this endeavor, we report a customized prime editing system for precision engineering of probiotic Escherichia coli Nissle 1917. With such a system, we developed a barcoding system for tracking engineered strains, and we built an antibiotic-resistance-gene-free platform to enable non-natural functionalities. We provide not only a powerful gene editing approach for probiotic bacteria but also new insights into the advancement of innovative CRISPR-Cas systems.},
}
RevDate: 2025-01-31
CmpDate: 2025-01-31
[A CRISPR/Cas approach to β-haemoglobinopathies].
Medecine sciences : M/S, 41(1):33-39.
Beta-haemoglobinopathies are severe genetic anemias caused by mutations that affect adult haemoglobin production. Many therapeutic approaches aim to reactivate the expression of the fetal hemoglobin genes. To this end, the CRISPR/Cas9 system has recently been used to genetically modify patients' hematopoietic stem/progenitor cells ex vivo and reactivate fetal hemoglobin expression in their erythroid progeny. More than 70 patients with severe β-thalassemia and sickle cell disease have been treated with the Casgevy® therapy. Most have achieved a significant improvement of clinical phenotype, with high editing efficiency in hematopoietic cells associated with normal or near normal hemoglobin levels. While the long-term safety and efficacy of this powerful approach still need to be evaluated, new strategies are being developed to further improve therapeutic outcomes, reduce potential genotoxicity and lower the costs of therapy.
Additional Links: PMID-39887096
Publisher:
PubMed:
Citation:
show bibtex listing
hide bibtex listing
@article {pmid39887096,
year = {2025},
author = {Brusson, M and Miccio, A},
title = {[A CRISPR/Cas approach to β-haemoglobinopathies].},
journal = {Medecine sciences : M/S},
volume = {41},
number = {1},
pages = {33-39},
doi = {10.1051/medsci/2024191},
pmid = {39887096},
issn = {1958-5381},
support = {ANR-10-IAHU-01//Agence nationale de la recherche/ ; 865797 DITSB//Conseil européen de la recherche/ ; subvention HORIZON-RIA EDITSCD n° 101057659//Commission européenne/ ; subvention 22206//AFM-Téléthon/ ; },
mesh = {Humans ; *CRISPR-Cas Systems ; *Gene Editing/methods ; *Genetic Therapy/methods ; *beta-Thalassemia/therapy/genetics ; Anemia, Sickle Cell/therapy/genetics ; Fetal Hemoglobin/genetics/biosynthesis ; Hematopoietic Stem Cells/metabolism/physiology ; Hemoglobinopathies/therapy/genetics ; Animals ; },
abstract = {Beta-haemoglobinopathies are severe genetic anemias caused by mutations that affect adult haemoglobin production. Many therapeutic approaches aim to reactivate the expression of the fetal hemoglobin genes. To this end, the CRISPR/Cas9 system has recently been used to genetically modify patients' hematopoietic stem/progenitor cells ex vivo and reactivate fetal hemoglobin expression in their erythroid progeny. More than 70 patients with severe β-thalassemia and sickle cell disease have been treated with the Casgevy® therapy. Most have achieved a significant improvement of clinical phenotype, with high editing efficiency in hematopoietic cells associated with normal or near normal hemoglobin levels. While the long-term safety and efficacy of this powerful approach still need to be evaluated, new strategies are being developed to further improve therapeutic outcomes, reduce potential genotoxicity and lower the costs of therapy.},
}
MeSH Terms:
show MeSH Terms
hide MeSH Terms
Humans
*CRISPR-Cas Systems
*Gene Editing/methods
*Genetic Therapy/methods
*beta-Thalassemia/therapy/genetics
Anemia, Sickle Cell/therapy/genetics
Fetal Hemoglobin/genetics/biosynthesis
Hematopoietic Stem Cells/metabolism/physiology
Hemoglobinopathies/therapy/genetics
Animals
RevDate: 2025-02-01
CmpDate: 2025-02-01
Differential roles of human CD4[+] and CD8[+] regulatory T cells in controlling self-reactive immune responses.
Nature immunology, 26(2):230-239.
Here we analyzed the relative contributions of CD4[+] regulatory T cells expressing Forkhead box protein P3 (FOXP3) and CD8[+] regulatory T cells expressing killer cell immunoglobulin-like receptors to the control of autoreactive T and B lymphocytes in human tonsil-derived immune organoids. FOXP3 and GZMB respectively encode proteins FOXP3 and granzyme B, which are critical to the suppressive functions of CD4[+] and CD8[+] regulatory T cells. Using CRISPR-Cas9 gene editing, we were able to achieve a reduction of ~90-95% in the expression of these genes. FOXP3 knockout in tonsil T cells led to production of antibodies against a variety of autoantigens and increased the affinity of influenza-specific antibodies. By contrast, GZMB knockout resulted in an increase in follicular helper T cells, consistent with the ablation of CD8[+] regulatory T cells observed in mouse models, and a marked expansion of autoreactive CD8[+] and CD4[+] T cells. These findings highlight the distinct yet complementary roles of CD8[+] and CD4[+] regulatory T cells in regulating cellular and humoral responses to prevent autoimmunity.
Additional Links: PMID-39806065
PubMed:
Citation:
show bibtex listing
hide bibtex listing
@article {pmid39806065,
year = {2025},
author = {Chen, X and Ghanizada, M and Mallajosyula, V and Sola, E and Capasso, R and Kathuria, KR and Davis, MM},
title = {Differential roles of human CD4[+] and CD8[+] regulatory T cells in controlling self-reactive immune responses.},
journal = {Nature immunology},
volume = {26},
number = {2},
pages = {230-239},
pmid = {39806065},
issn = {1529-2916},
support = {U19 AI057229/AI/NIAID NIH HHS/United States ; AI057229//Division of Intramural Research, National Institute of Allergy and Infectious Diseases (Division of Intramural Research of the NIAID)/ ; },
mesh = {Humans ; *T-Lymphocytes, Regulatory/immunology ; *CD8-Positive T-Lymphocytes/immunology ; *Forkhead Transcription Factors/metabolism ; *Granzymes/metabolism ; *Autoimmunity ; Palatine Tonsil/immunology ; Animals ; Autoantigens/immunology ; Mice ; CRISPR-Cas Systems ; },
abstract = {Here we analyzed the relative contributions of CD4[+] regulatory T cells expressing Forkhead box protein P3 (FOXP3) and CD8[+] regulatory T cells expressing killer cell immunoglobulin-like receptors to the control of autoreactive T and B lymphocytes in human tonsil-derived immune organoids. FOXP3 and GZMB respectively encode proteins FOXP3 and granzyme B, which are critical to the suppressive functions of CD4[+] and CD8[+] regulatory T cells. Using CRISPR-Cas9 gene editing, we were able to achieve a reduction of ~90-95% in the expression of these genes. FOXP3 knockout in tonsil T cells led to production of antibodies against a variety of autoantigens and increased the affinity of influenza-specific antibodies. By contrast, GZMB knockout resulted in an increase in follicular helper T cells, consistent with the ablation of CD8[+] regulatory T cells observed in mouse models, and a marked expansion of autoreactive CD8[+] and CD4[+] T cells. These findings highlight the distinct yet complementary roles of CD8[+] and CD4[+] regulatory T cells in regulating cellular and humoral responses to prevent autoimmunity.},
}
MeSH Terms:
show MeSH Terms
hide MeSH Terms
Humans
*T-Lymphocytes, Regulatory/immunology
*CD8-Positive T-Lymphocytes/immunology
*Forkhead Transcription Factors/metabolism
*Granzymes/metabolism
*Autoimmunity
Palatine Tonsil/immunology
Animals
Autoantigens/immunology
Mice
CRISPR-Cas Systems
RevDate: 2025-01-31
CmpDate: 2025-01-31
Non-viral gene therapy for Leber's congenital amaurosis: progress and possibilities.
Nanomedicine (London, England), 20(3):291-304.
Leber's congenital amaurosis (LCA) represents a set of rare and pervasive hereditary conditions of the retina that cause severe vision loss starting in early childhood. Targeted treatment intervention has become possible thanks to recent advances in understanding LCA genetic basis. While viral vectors have shown efficacy in gene delivery, they present challenges related to safety, low cargo capacity, and the potential for random genomic integration. Non-viral gene therapy is a safer and more flexible alternative to treating the underlying genetic mutation causing LCA. Non-viral gene delivery methods, such as inorganic nanoparticles, polymer-based delivery systems, and lipid-based nanoparticles, bypass the risks of immunogenicity and genomic integration, potentially offering a more versatile and personalized treatment for patients. This review explores the genetic background of LCA, emphasizing the mutations involved, and explores diverse non-viral gene delivery methods being developed. It also highlights recent studies on non-viral gene therapy for LCA in animal models and clinical trials. It presents future perspectives for gene therapy, including integrating emerging technologies like CRISPR-Cas9, interdisciplinary collaborations, personalized medicine, and ethical considerations.
Additional Links: PMID-39707712
Publisher:
PubMed:
Citation:
show bibtex listing
hide bibtex listing
@article {pmid39707712,
year = {2025},
author = {Abdulsalam, L and Mordecai, J and Ahmad, I},
title = {Non-viral gene therapy for Leber's congenital amaurosis: progress and possibilities.},
journal = {Nanomedicine (London, England)},
volume = {20},
number = {3},
pages = {291-304},
doi = {10.1080/17435889.2024.2443387},
pmid = {39707712},
issn = {1748-6963},
mesh = {Humans ; *Genetic Therapy/methods ; *Leber Congenital Amaurosis/therapy/genetics ; Animals ; *Gene Transfer Techniques ; *Nanoparticles/chemistry ; Genetic Vectors/genetics ; Mutation ; CRISPR-Cas Systems/genetics ; },
abstract = {Leber's congenital amaurosis (LCA) represents a set of rare and pervasive hereditary conditions of the retina that cause severe vision loss starting in early childhood. Targeted treatment intervention has become possible thanks to recent advances in understanding LCA genetic basis. While viral vectors have shown efficacy in gene delivery, they present challenges related to safety, low cargo capacity, and the potential for random genomic integration. Non-viral gene therapy is a safer and more flexible alternative to treating the underlying genetic mutation causing LCA. Non-viral gene delivery methods, such as inorganic nanoparticles, polymer-based delivery systems, and lipid-based nanoparticles, bypass the risks of immunogenicity and genomic integration, potentially offering a more versatile and personalized treatment for patients. This review explores the genetic background of LCA, emphasizing the mutations involved, and explores diverse non-viral gene delivery methods being developed. It also highlights recent studies on non-viral gene therapy for LCA in animal models and clinical trials. It presents future perspectives for gene therapy, including integrating emerging technologies like CRISPR-Cas9, interdisciplinary collaborations, personalized medicine, and ethical considerations.},
}
MeSH Terms:
show MeSH Terms
hide MeSH Terms
Humans
*Genetic Therapy/methods
*Leber Congenital Amaurosis/therapy/genetics
Animals
*Gene Transfer Techniques
*Nanoparticles/chemistry
Genetic Vectors/genetics
Mutation
CRISPR-Cas Systems/genetics
RevDate: 2025-02-01
CmpDate: 2025-02-01
Induced pluripotent stem cell-derived macrophages as a platform for modelling human disease.
Nature reviews. Immunology, 25(2):108-124.
Macrophages are innate immune cells that are present in essentially all tissues, where they have vital roles in tissue development, homeostasis and pathogenesis. The importance of macrophages in tissue function is reflected by their association with various human diseases, and studying macrophage functions in both homeostasis and pathological tissue settings is a promising avenue for new targeted therapies that will improve human health. The ability to generate macrophages from induced pluripotent stem (iPS) cells has revolutionized macrophage biology, with the generation of iPS cell-derived macrophages (iMacs) providing unlimited access to genotype-specific cells that can be used to model various human diseases involving macrophage dysregulation. Such disease modelling is achieved by generating iPS cells from patient-derived cells carrying disease-related mutations or by introducing mutations into iPS cells from healthy donors using CRISPR-Cas9 technology. These iMacs that carry disease-related mutations can be used to study the aetiology of the particular disease in vitro. To achieve more physiological relevance, iMacs can be co-cultured in 2D systems with iPS cell-derived cells or in 3D systems with iPS cell-derived organoids. Here, we discuss the studies that have attempted to model various human diseases using iMacs, highlighting how these have advanced our knowledge about the role of macrophages in health and disease.
Additional Links: PMID-39333753
PubMed:
Citation:
show bibtex listing
hide bibtex listing
@article {pmid39333753,
year = {2025},
author = {Tiwari, SK and Wong, WJ and Moreira, M and Pasqualini, C and Ginhoux, F},
title = {Induced pluripotent stem cell-derived macrophages as a platform for modelling human disease.},
journal = {Nature reviews. Immunology},
volume = {25},
number = {2},
pages = {108-124},
pmid = {39333753},
issn = {1474-1741},
mesh = {Humans ; *Induced Pluripotent Stem Cells/immunology/cytology ; *Macrophages/immunology ; *Cell Differentiation/immunology ; Animals ; CRISPR-Cas Systems ; Organoids/immunology ; },
abstract = {Macrophages are innate immune cells that are present in essentially all tissues, where they have vital roles in tissue development, homeostasis and pathogenesis. The importance of macrophages in tissue function is reflected by their association with various human diseases, and studying macrophage functions in both homeostasis and pathological tissue settings is a promising avenue for new targeted therapies that will improve human health. The ability to generate macrophages from induced pluripotent stem (iPS) cells has revolutionized macrophage biology, with the generation of iPS cell-derived macrophages (iMacs) providing unlimited access to genotype-specific cells that can be used to model various human diseases involving macrophage dysregulation. Such disease modelling is achieved by generating iPS cells from patient-derived cells carrying disease-related mutations or by introducing mutations into iPS cells from healthy donors using CRISPR-Cas9 technology. These iMacs that carry disease-related mutations can be used to study the aetiology of the particular disease in vitro. To achieve more physiological relevance, iMacs can be co-cultured in 2D systems with iPS cell-derived cells or in 3D systems with iPS cell-derived organoids. Here, we discuss the studies that have attempted to model various human diseases using iMacs, highlighting how these have advanced our knowledge about the role of macrophages in health and disease.},
}
MeSH Terms:
show MeSH Terms
hide MeSH Terms
Humans
*Induced Pluripotent Stem Cells/immunology/cytology
*Macrophages/immunology
*Cell Differentiation/immunology
Animals
CRISPR-Cas Systems
Organoids/immunology
RevDate: 2025-02-01
CmpDate: 2025-02-01
Gene drives: an alternative approach to malaria control?.
Gene therapy, 32(1):25-37.
Genetic modification for the control of mosquitoes is frequently touted as a solution for a variety of vector-borne diseases. There has been some success using non-insecticidal methods like sterile or incompatible insect techniques to control arbovirus diseases. However, control by genetic modifications to reduce mosquito populations or create mosquitoes that are refractory to infection with pathogens are less developed. The advent of CRISPR-Cas9-mediated gene drives may advance this mechanism of control. In this review, use and progress of gene drives for vector control, particularly for malaria, is discussed. A brief history of population suppression and replacement gene drives in mosquitoes, rapid advancement of the field over the last decade and how genetic modification fits into the current scope of vector control are described. Mechanisms of alternative vector control by genetic modification to modulate mosquitoes' immune responses and anti-parasite effector molecules as part of a combinational strategy to combat malaria are considered. Finally, the limitations and ethics of using gene drives for mosquito control are discussed.
Additional Links: PMID-39039203
PubMed:
Citation:
show bibtex listing
hide bibtex listing
@article {pmid39039203,
year = {2025},
author = {Naidoo, K and Oliver, SV},
title = {Gene drives: an alternative approach to malaria control?.},
journal = {Gene therapy},
volume = {32},
number = {1},
pages = {25-37},
pmid = {39039203},
issn = {1476-5462},
mesh = {*Malaria/prevention & control ; Animals ; *Gene Drive Technology/methods ; Humans ; *Mosquito Vectors/genetics ; *CRISPR-Cas Systems ; *Mosquito Control/methods ; Gene Editing/methods ; Culicidae/genetics ; },
abstract = {Genetic modification for the control of mosquitoes is frequently touted as a solution for a variety of vector-borne diseases. There has been some success using non-insecticidal methods like sterile or incompatible insect techniques to control arbovirus diseases. However, control by genetic modifications to reduce mosquito populations or create mosquitoes that are refractory to infection with pathogens are less developed. The advent of CRISPR-Cas9-mediated gene drives may advance this mechanism of control. In this review, use and progress of gene drives for vector control, particularly for malaria, is discussed. A brief history of population suppression and replacement gene drives in mosquitoes, rapid advancement of the field over the last decade and how genetic modification fits into the current scope of vector control are described. Mechanisms of alternative vector control by genetic modification to modulate mosquitoes' immune responses and anti-parasite effector molecules as part of a combinational strategy to combat malaria are considered. Finally, the limitations and ethics of using gene drives for mosquito control are discussed.},
}
MeSH Terms:
show MeSH Terms
hide MeSH Terms
*Malaria/prevention & control
Animals
*Gene Drive Technology/methods
Humans
*Mosquito Vectors/genetics
*CRISPR-Cas Systems
*Mosquito Control/methods
Gene Editing/methods
Culicidae/genetics
RevDate: 2025-01-30
CmpDate: 2025-01-31
Intricate interplay of CRISPR-Cas systems, anti-CRISPR proteins, and antimicrobial resistance genes in a globally successful multi-drug resistant Klebsiella pneumoniae clone.
Genome medicine, 17(1):9.
BACKGROUND: Klebsiella pneumoniae is one of the most prevalent pathogens responsible for multiple infections in healthcare settings and the community. K. pneumoniae CG147, primarily including ST147 (the founder ST), ST273, and ST392, is one of the most globally successful MDR clone linked to various carbapenemases.
METHODS: One hundred and one CG147 strains were sequenced and additional 911 publicly available CG147 genome sequences were included for analysis. The molecular epidemiology, population structure, and time phylogeny were investigated. The virulome, resistome, and mobilome were analyzed, and the recombination in the capsular region was studied. The CRISPR-Cas and anti-CRISPR were identified. The interplay between CRISPR-Cas, anti-CRISPR, and carbapenemase-encoding plasmids was analyzed and experimentally validated.
RESULTS: We analyzed 1012 global CG147 genomes, with 80.4% encoding at least one carbapenemase (NDM [529/1012, 52.3%], OXA-48-like [182/1012, 17.7%], and KPC [105/1012, 10.4%]). Surprisingly, almost all CG147 strains (99.7%, 1009/1,012) harbor a chromosomal type I-E CRISPR-Cas system, with 41.8% (423/1012) containing an additional plasmid-borne type IV-A3 CRISPR-Cas system, and both target IncF plasmids, e.g., the most prevalent KPC-encoding pKpQIL-like plasmids. We found the presence of IV-A3 CRISPR-Cas system showed a negative correlation with the presence of KPC. Interestingly, a prophage-encoding anti-CRISPR AcrIE8.1 and a plasmid-borne anti-CRISPR AcrIE9.2 were detected in 40.1% (406/1012) and 54.2% (548/1012) of strains, respectively, which displayed positive correlations with the presence of a carbapenemase. Plasmid transfer experiments confirmed that the I-E and IV-A3 CRISPR-Cas systems significantly decreased (p < 0.001) KPC-encoding pKpQIL plasmid conjugation frequencies, while the AcrIE8.1 and AcrIE9.2 significantly increased (p < 0.001) pKpQIL conjugation frequencies and protected plasmids from elimination by CRISPR-Cas I-E system.
CONCLUSIONS: Our results indicated a complex interplay between CRISPR-Cas, anti-CRISPR, and mobile genetic elements that shape the evolution of CG147. Our findings advance the understanding of multi-drug resistance mechanisms and will aid in preventing the emergence of future MDR clones.
Additional Links: PMID-39885543
PubMed:
Citation:
show bibtex listing
hide bibtex listing
@article {pmid39885543,
year = {2025},
author = {Jiang, J and Cienfuegos-Galletd, AV and Long, T and Peirano, G and Chu, T and Pitout, JDD and Kreiswirth, BN and Chen, L},
title = {Intricate interplay of CRISPR-Cas systems, anti-CRISPR proteins, and antimicrobial resistance genes in a globally successful multi-drug resistant Klebsiella pneumoniae clone.},
journal = {Genome medicine},
volume = {17},
number = {1},
pages = {9},
pmid = {39885543},
issn = {1756-994X},
mesh = {*Klebsiella pneumoniae/genetics/drug effects ; *CRISPR-Cas Systems ; *Drug Resistance, Multiple, Bacterial/genetics ; *Bacterial Proteins/genetics ; Phylogeny ; Plasmids/genetics ; beta-Lactamases/genetics ; Genome, Bacterial ; Klebsiella Infections/microbiology ; Humans ; Anti-Bacterial Agents/pharmacology ; },
abstract = {BACKGROUND: Klebsiella pneumoniae is one of the most prevalent pathogens responsible for multiple infections in healthcare settings and the community. K. pneumoniae CG147, primarily including ST147 (the founder ST), ST273, and ST392, is one of the most globally successful MDR clone linked to various carbapenemases.
METHODS: One hundred and one CG147 strains were sequenced and additional 911 publicly available CG147 genome sequences were included for analysis. The molecular epidemiology, population structure, and time phylogeny were investigated. The virulome, resistome, and mobilome were analyzed, and the recombination in the capsular region was studied. The CRISPR-Cas and anti-CRISPR were identified. The interplay between CRISPR-Cas, anti-CRISPR, and carbapenemase-encoding plasmids was analyzed and experimentally validated.
RESULTS: We analyzed 1012 global CG147 genomes, with 80.4% encoding at least one carbapenemase (NDM [529/1012, 52.3%], OXA-48-like [182/1012, 17.7%], and KPC [105/1012, 10.4%]). Surprisingly, almost all CG147 strains (99.7%, 1009/1,012) harbor a chromosomal type I-E CRISPR-Cas system, with 41.8% (423/1012) containing an additional plasmid-borne type IV-A3 CRISPR-Cas system, and both target IncF plasmids, e.g., the most prevalent KPC-encoding pKpQIL-like plasmids. We found the presence of IV-A3 CRISPR-Cas system showed a negative correlation with the presence of KPC. Interestingly, a prophage-encoding anti-CRISPR AcrIE8.1 and a plasmid-borne anti-CRISPR AcrIE9.2 were detected in 40.1% (406/1012) and 54.2% (548/1012) of strains, respectively, which displayed positive correlations with the presence of a carbapenemase. Plasmid transfer experiments confirmed that the I-E and IV-A3 CRISPR-Cas systems significantly decreased (p < 0.001) KPC-encoding pKpQIL plasmid conjugation frequencies, while the AcrIE8.1 and AcrIE9.2 significantly increased (p < 0.001) pKpQIL conjugation frequencies and protected plasmids from elimination by CRISPR-Cas I-E system.
CONCLUSIONS: Our results indicated a complex interplay between CRISPR-Cas, anti-CRISPR, and mobile genetic elements that shape the evolution of CG147. Our findings advance the understanding of multi-drug resistance mechanisms and will aid in preventing the emergence of future MDR clones.},
}
MeSH Terms:
show MeSH Terms
hide MeSH Terms
*Klebsiella pneumoniae/genetics/drug effects
*CRISPR-Cas Systems
*Drug Resistance, Multiple, Bacterial/genetics
*Bacterial Proteins/genetics
Phylogeny
Plasmids/genetics
beta-Lactamases/genetics
Genome, Bacterial
Klebsiella Infections/microbiology
Humans
Anti-Bacterial Agents/pharmacology
RevDate: 2025-01-30
CmpDate: 2025-01-31
PathoGD: an integrative genomics approach to primer and guide RNA design for CRISPR-based diagnostics.
Communications biology, 8(1):147.
Critical to the success of CRISPR-based diagnostic assays is the selection of a diagnostic target highly specific to the organism of interest, a process often requiring iterative cycles of manual selection, optimisation, and redesign. Here we present PathoGD, a bioinformatic pipeline for rapid and high-throughput design of RPA primers and gRNAs for CRISPR-Cas12a-based pathogen detection. PathoGD is fully automated, leverages publicly available sequences and is scalable to large datasets, allowing rapid continuous monitoring and validation of primer/gRNA sets to ensure ongoing assay relevance. We designed primers and gRNAs for five clinically relevant bacterial pathogens, and experimentally validated a subset of the designs for detecting Streptococcus pyogenes and/or Neisseria gonorrhoeae in assays with and without pre-amplification. We demonstrated high specificity of primers and gRNAs designed, with minimal off-target signal observed for all combinations. We anticipate PathoGD will be an important resource for assay design for current and emerging pathogens. PathoGD is available on GitHub at https://github.com/sjlow23/pathogd .
Additional Links: PMID-39885339
PubMed:
Citation:
show bibtex listing
hide bibtex listing
@article {pmid39885339,
year = {2025},
author = {Low, SJ and O'Neill, M and Kerry, WJ and Wild, N and Krysiak, M and Nong, Y and Azzato, F and Hor, E and Williams, L and Taiaroa, G and Steinig, E and Pasricha, S and Williamson, DA},
title = {PathoGD: an integrative genomics approach to primer and guide RNA design for CRISPR-based diagnostics.},
journal = {Communications biology},
volume = {8},
number = {1},
pages = {147},
pmid = {39885339},
issn = {2399-3642},
support = {GA-F3791196-5514//State Government of Victoria (Victorian Government)/ ; PO4932//Department of Health, Australian Government (Department of Health)/ ; },
mesh = {*Genomics/methods ; *Streptococcus pyogenes/genetics ; *CRISPR-Cas Systems ; *RNA, Guide, CRISPR-Cas Systems/genetics ; *Neisseria gonorrhoeae/genetics/isolation & purification ; Humans ; DNA Primers/genetics ; Computational Biology/methods ; Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {Critical to the success of CRISPR-based diagnostic assays is the selection of a diagnostic target highly specific to the organism of interest, a process often requiring iterative cycles of manual selection, optimisation, and redesign. Here we present PathoGD, a bioinformatic pipeline for rapid and high-throughput design of RPA primers and gRNAs for CRISPR-Cas12a-based pathogen detection. PathoGD is fully automated, leverages publicly available sequences and is scalable to large datasets, allowing rapid continuous monitoring and validation of primer/gRNA sets to ensure ongoing assay relevance. We designed primers and gRNAs for five clinically relevant bacterial pathogens, and experimentally validated a subset of the designs for detecting Streptococcus pyogenes and/or Neisseria gonorrhoeae in assays with and without pre-amplification. We demonstrated high specificity of primers and gRNAs designed, with minimal off-target signal observed for all combinations. We anticipate PathoGD will be an important resource for assay design for current and emerging pathogens. PathoGD is available on GitHub at https://github.com/sjlow23/pathogd .},
}
MeSH Terms:
show MeSH Terms
hide MeSH Terms
*Genomics/methods
*Streptococcus pyogenes/genetics
*CRISPR-Cas Systems
*RNA, Guide, CRISPR-Cas Systems/genetics
*Neisseria gonorrhoeae/genetics/isolation & purification
Humans
DNA Primers/genetics
Computational Biology/methods
Clustered Regularly Interspaced Short Palindromic Repeats
RevDate: 2025-01-30
CmpDate: 2025-01-31
Tunable control of Cas12 activity promotes universal and fast one-pot nucleic acid detection.
Nature communications, 16(1):1166.
The CRISPR-based detection methods have been widely applied, yet they remain limited by the non-universal nature of one-pot diagnostic approaches. Here, we report a universal one-pot fluorescent method for the detection of epidemic pathogens, delivering results within 15-20 min. This method uses heparin sodium to precisely tunes the cis-cleavage capability of Cas12 via interference with the Cas12a-crRNA binding process, thereby generating significant fluorescence due to the accumulation of isothermal amplification products. Additionally, this universal assay accommodates both classic and suboptimal PAMs, as well as various Cas12a subtypes such as LbCas12a, AsCas12a, and AapCas12b. Such a robust method demonstrates sensitivity and specificity exceeding 95% in the detection of monkeypox pseudovirus, influenza A virus, and SARS-CoV-2 from saliva or wastewater samples, when compared with qPCR or RT-qPCR. Moreover, the cost of heparin sodium per thousand uses is $0.01 to $0.04 only. Collectively, this universal and fast one-pot approach based on heparin sodium offers potential possibilities for point-of-care testing.
Additional Links: PMID-39885211
PubMed:
Citation:
show bibtex listing
hide bibtex listing
@article {pmid39885211,
year = {2025},
author = {Cheng, ZH and Luo, XY and Yu, SS and Min, D and Zhang, SX and Li, XF and Chen, JJ and Liu, DF and Yu, HQ},
title = {Tunable control of Cas12 activity promotes universal and fast one-pot nucleic acid detection.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {1166},
pmid = {39885211},
issn = {2041-1723},
mesh = {*SARS-CoV-2/genetics/isolation & purification ; *CRISPR-Cas Systems ; Humans ; Nucleic Acid Amplification Techniques/methods ; CRISPR-Associated Proteins/metabolism/genetics ; Influenza A virus/genetics ; COVID-19/virology/diagnosis ; Bacterial Proteins/genetics/metabolism ; Saliva/virology ; Endodeoxyribonucleases/metabolism/genetics ; Sensitivity and Specificity ; Heparin/metabolism ; Molecular Diagnostic Techniques/methods ; },
abstract = {The CRISPR-based detection methods have been widely applied, yet they remain limited by the non-universal nature of one-pot diagnostic approaches. Here, we report a universal one-pot fluorescent method for the detection of epidemic pathogens, delivering results within 15-20 min. This method uses heparin sodium to precisely tunes the cis-cleavage capability of Cas12 via interference with the Cas12a-crRNA binding process, thereby generating significant fluorescence due to the accumulation of isothermal amplification products. Additionally, this universal assay accommodates both classic and suboptimal PAMs, as well as various Cas12a subtypes such as LbCas12a, AsCas12a, and AapCas12b. Such a robust method demonstrates sensitivity and specificity exceeding 95% in the detection of monkeypox pseudovirus, influenza A virus, and SARS-CoV-2 from saliva or wastewater samples, when compared with qPCR or RT-qPCR. Moreover, the cost of heparin sodium per thousand uses is $0.01 to $0.04 only. Collectively, this universal and fast one-pot approach based on heparin sodium offers potential possibilities for point-of-care testing.},
}
MeSH Terms:
show MeSH Terms
hide MeSH Terms
*SARS-CoV-2/genetics/isolation & purification
*CRISPR-Cas Systems
Humans
Nucleic Acid Amplification Techniques/methods
CRISPR-Associated Proteins/metabolism/genetics
Influenza A virus/genetics
COVID-19/virology/diagnosis
Bacterial Proteins/genetics/metabolism
Saliva/virology
Endodeoxyribonucleases/metabolism/genetics
Sensitivity and Specificity
Heparin/metabolism
Molecular Diagnostic Techniques/methods
RevDate: 2025-01-30
CmpDate: 2025-01-30
Advancing the genetic engineering toolbox by combining AsCas12a knock-in mice with ultra-compact screening.
Nature communications, 16(1):974.
Cas12a is a next-generation gene editing tool that enables multiplexed gene targeting. Here, we present a mouse model that constitutively expresses enhanced Acidaminococcus sp. Cas12a (enAsCas12a) linked to an mCherry fluorescent reporter. We demonstrate efficient single and multiplexed gene editing in vitro, using primary and transformed cells from enAsCas12a mice. We further demonstrate successful in vivo gene editing, using normal and cancer-prone enAsCas12a stem cells to reconstitute the haematopoietic system of wild-type mice. We also present compact, genome-wide Cas12a knockout libraries, with four crRNAs per gene encoded across one (Scherzo) or two (Menuetto) vectors, and demonstrate the utility of these libraries across methodologies: in vitro enrichment and drop-out screening in lymphoma cells and immortalised fibroblasts, respectively, and in vivo screens to identify lymphoma-driving events. Finally, we demonstrate CRISPR multiplexing via simultaneous gene knockout (via Cas12a) and activation (via dCas9-SAM) using primary T cells and fibroblasts. Our enAsCas12a mouse and accompanying crRNA libraries enhance genome engineering capabilities and complement current CRISPR technologies.
Additional Links: PMID-39885149
PubMed:
Citation:
show bibtex listing
hide bibtex listing
@article {pmid39885149,
year = {2025},
author = {Jin, W and Deng, Y and La Marca, JE and Lelliott, EJ and Diepstraten, ST and König, C and Tai, L and Snetkova, V and Dorighi, KM and Hoberecht, L and Hedditch, MG and Whelan, L and Healey, G and Fayle, D and Lau, K and Potts, MA and Chen, MZ and Johnston, APR and Liao, Y and Shi, W and Kueh, AJ and Haley, B and Fortin, JP and Herold, MJ},
title = {Advancing the genetic engineering toolbox by combining AsCas12a knock-in mice with ultra-compact screening.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {974},
pmid = {39885149},
issn = {2041-1723},
mesh = {Animals ; Mice ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Gene Knock-In Techniques/methods ; CRISPR-Associated Proteins/genetics/metabolism ; Lymphoma/genetics ; Genetic Engineering/methods ; Bacterial Proteins/genetics/metabolism ; Endodeoxyribonucleases/genetics/metabolism ; Humans ; Gene Knockout Techniques/methods ; Red Fluorescent Protein ; Fibroblasts/metabolism ; Mice, Inbred C57BL ; },
abstract = {Cas12a is a next-generation gene editing tool that enables multiplexed gene targeting. Here, we present a mouse model that constitutively expresses enhanced Acidaminococcus sp. Cas12a (enAsCas12a) linked to an mCherry fluorescent reporter. We demonstrate efficient single and multiplexed gene editing in vitro, using primary and transformed cells from enAsCas12a mice. We further demonstrate successful in vivo gene editing, using normal and cancer-prone enAsCas12a stem cells to reconstitute the haematopoietic system of wild-type mice. We also present compact, genome-wide Cas12a knockout libraries, with four crRNAs per gene encoded across one (Scherzo) or two (Menuetto) vectors, and demonstrate the utility of these libraries across methodologies: in vitro enrichment and drop-out screening in lymphoma cells and immortalised fibroblasts, respectively, and in vivo screens to identify lymphoma-driving events. Finally, we demonstrate CRISPR multiplexing via simultaneous gene knockout (via Cas12a) and activation (via dCas9-SAM) using primary T cells and fibroblasts. Our enAsCas12a mouse and accompanying crRNA libraries enhance genome engineering capabilities and complement current CRISPR technologies.},
}
MeSH Terms:
show MeSH Terms
hide MeSH Terms
Animals
Mice
*CRISPR-Cas Systems/genetics
*Gene Editing/methods
Gene Knock-In Techniques/methods
CRISPR-Associated Proteins/genetics/metabolism
Lymphoma/genetics
Genetic Engineering/methods
Bacterial Proteins/genetics/metabolism
Endodeoxyribonucleases/genetics/metabolism
Humans
Gene Knockout Techniques/methods
Red Fluorescent Protein
Fibroblasts/metabolism
Mice, Inbred C57BL
RevDate: 2025-01-31
CmpDate: 2025-01-31
Dual α-globin-truncated erythropoietin receptor knockin restores hemoglobin production in α-thalassemia-derived erythroid cells.
Cell reports, 44(1):115141.
The most severe form of α-thalassemia results from loss of all four copies of α-globin. Postnatally, patients face challenges similar to β-thalassemia, including severe anemia and erythrotoxicity due to the imbalance of β-globin and α-globin chains. Despite progress in genome editing treatments for β-thalassemia, there is no analogous curative option for α-thalassemia. To address this, we designed a Cas9/AAV6-mediated genome editing strategy that integrates a functional α-globin gene into the β-globin locus in α-thalassemia patient-derived hematopoietic stem and progenitor cells (HSPCs). Incorporation of a truncated erythropoietin receptor transgene into the α-globin integration cassette significantly increased erythropoietic output from edited HSPCs and led to the most robust production of α-globin, and consequently hemoglobin tetramers. By directing edited HSPCs toward increased production of clinically relevant erythroid cells, this approach has the potential to mitigate the limitations of current treatments for the hemoglobinopathies, including low genome editing and low engraftment rates.
Additional Links: PMID-39754719
Publisher:
PubMed:
Citation:
show bibtex listing
hide bibtex listing
@article {pmid39754719,
year = {2025},
author = {Chu, SN and Soupene, E and Sharma, D and Sinha, R and McCreary, T and Hernandez, B and Shen, H and Wienert, B and Bowman, C and Yin, H and Lesch, BJ and Jia, K and Romero, KA and Kostamo, Z and Zhang, Y and Tran, T and Cordero, M and Homma, S and Hampton, JP and Gardner, JM and Conklin, BR and MacKenzie, TC and Sheehan, VA and Porteus, MH and Cromer, MK},
title = {Dual α-globin-truncated erythropoietin receptor knockin restores hemoglobin production in α-thalassemia-derived erythroid cells.},
journal = {Cell reports},
volume = {44},
number = {1},
pages = {115141},
doi = {10.1016/j.celrep.2024.115141},
pmid = {39754719},
issn = {2211-1247},
support = {T32 AI125222/AI/NIAID NIH HHS/United States ; },
mesh = {Humans ; *alpha-Globins/genetics/metabolism ; *Erythroid Cells/metabolism ; *alpha-Thalassemia/genetics/metabolism ; *Receptors, Erythropoietin/metabolism/genetics ; *Gene Editing/methods ; *Hemoglobins/metabolism ; Gene Knock-In Techniques ; CRISPR-Cas Systems/genetics ; Erythropoiesis/genetics ; Hematopoietic Stem Cells/metabolism ; beta-Globins/genetics/metabolism ; },
abstract = {The most severe form of α-thalassemia results from loss of all four copies of α-globin. Postnatally, patients face challenges similar to β-thalassemia, including severe anemia and erythrotoxicity due to the imbalance of β-globin and α-globin chains. Despite progress in genome editing treatments for β-thalassemia, there is no analogous curative option for α-thalassemia. To address this, we designed a Cas9/AAV6-mediated genome editing strategy that integrates a functional α-globin gene into the β-globin locus in α-thalassemia patient-derived hematopoietic stem and progenitor cells (HSPCs). Incorporation of a truncated erythropoietin receptor transgene into the α-globin integration cassette significantly increased erythropoietic output from edited HSPCs and led to the most robust production of α-globin, and consequently hemoglobin tetramers. By directing edited HSPCs toward increased production of clinically relevant erythroid cells, this approach has the potential to mitigate the limitations of current treatments for the hemoglobinopathies, including low genome editing and low engraftment rates.},
}
MeSH Terms:
show MeSH Terms
hide MeSH Terms
Humans
*alpha-Globins/genetics/metabolism
*Erythroid Cells/metabolism
*alpha-Thalassemia/genetics/metabolism
*Receptors, Erythropoietin/metabolism/genetics
*Gene Editing/methods
*Hemoglobins/metabolism
Gene Knock-In Techniques
CRISPR-Cas Systems/genetics
Erythropoiesis/genetics
Hematopoietic Stem Cells/metabolism
beta-Globins/genetics/metabolism
RevDate: 2025-01-31
CmpDate: 2025-01-31
Improved split prime editors enable efficient in vivo genome editing.
Cell reports, 44(1):115144.
Efficient prime editor (PE) delivery in vivo is critical for realizing its full potential in disease modeling and therapeutic correction. Although PE has been divided into two halves and delivered using dual adeno-associated viruses (AAVs), the editing efficiency at different gene loci varies among split sites. Furthermore, efficient split sites within Cas9 nickase (Cas9n) are limited. Here, we verified that 1115 (Asn) is an efficient split site when delivering PEs by dual AAVs. Additionally, we utilized a feature in which reverse transcriptase could be detached from the Cas9n and designed split sites in the first half of Cas9n. We found that split-PE-367 enabled high editing efficiency with Rma intein. To test the editing efficiency in vivo, split-ePE3-367 was packaged in AAV9 and achieved 17.5% precise editing in mice. Our findings establish an alternative split-PE architecture that enables robust editing efficiency, facilitating potential utility in disease modeling and correction.
Additional Links: PMID-39745853
Publisher:
PubMed:
Citation:
show bibtex listing
hide bibtex listing
@article {pmid39745853,
year = {2025},
author = {Wei, R and Yu, Z and Ding, L and Lu, Z and Yao, K and Zhang, H and Huang, B and He, M and Ma, L},
title = {Improved split prime editors enable efficient in vivo genome editing.},
journal = {Cell reports},
volume = {44},
number = {1},
pages = {115144},
doi = {10.1016/j.celrep.2024.115144},
pmid = {39745853},
issn = {2211-1247},
mesh = {*Gene Editing/methods ; Animals ; *Dependovirus/genetics ; Mice ; Humans ; CRISPR-Cas Systems/genetics ; HEK293 Cells ; },
abstract = {Efficient prime editor (PE) delivery in vivo is critical for realizing its full potential in disease modeling and therapeutic correction. Although PE has been divided into two halves and delivered using dual adeno-associated viruses (AAVs), the editing efficiency at different gene loci varies among split sites. Furthermore, efficient split sites within Cas9 nickase (Cas9n) are limited. Here, we verified that 1115 (Asn) is an efficient split site when delivering PEs by dual AAVs. Additionally, we utilized a feature in which reverse transcriptase could be detached from the Cas9n and designed split sites in the first half of Cas9n. We found that split-PE-367 enabled high editing efficiency with Rma intein. To test the editing efficiency in vivo, split-ePE3-367 was packaged in AAV9 and achieved 17.5% precise editing in mice. Our findings establish an alternative split-PE architecture that enables robust editing efficiency, facilitating potential utility in disease modeling and correction.},
}
MeSH Terms:
show MeSH Terms
hide MeSH Terms
*Gene Editing/methods
Animals
*Dependovirus/genetics
Mice
Humans
CRISPR-Cas Systems/genetics
HEK293 Cells
RevDate: 2025-01-31
CmpDate: 2025-01-31
Dual-nuclease single-cell lineage tracing by Cas9 and Cas12a.
Cell reports, 44(1):115105.
Single-cell lineage tracing based on CRISPR-Cas9 gene editing enables the simultaneous linkage of cell states and lineage history at a high resolution. Despite its immense potential in resolving the cell fate determination and genealogy within an organism, existing implementations of this technology suffer from limitations in recording capabilities and considerable barcode dropout. Here, we introduce DuTracer, a versatile tool that utilizes two orthogonal gene editing systems to record cell lineage history at single-cell resolution in an inducible manner. DuTracer shows the ability to enhance lineage recording with minimized target dropouts and potentially deeper tree depths. Applying DuTracer in mouse embryoid bodies and neuromesodermal organoids illustrates the lineage relationship of different cell types and proposes potential lineage-biased molecular drivers, showcased by identifying transcription factor Foxb1 as a modulator in the cell fate determination of neuromesodermal progenitors. Collectively, DuTracer facilitates the precise and regulatory interrogation of cellular lineages of complex biological processes.
Additional Links: PMID-39721023
Publisher:
PubMed:
Citation:
show bibtex listing
hide bibtex listing
@article {pmid39721023,
year = {2025},
author = {Chen, C and Liao, Y and Zhu, M and Wang, L and Yu, X and Li, M and Peng, G},
title = {Dual-nuclease single-cell lineage tracing by Cas9 and Cas12a.},
journal = {Cell reports},
volume = {44},
number = {1},
pages = {115105},
doi = {10.1016/j.celrep.2024.115105},
pmid = {39721023},
issn = {2211-1247},
mesh = {Animals ; *Cell Lineage ; Mice ; *Single-Cell Analysis/methods ; *CRISPR-Cas Systems/genetics ; Gene Editing/methods ; CRISPR-Associated Protein 9/metabolism ; CRISPR-Associated Proteins/metabolism/genetics ; Cell Differentiation ; Endodeoxyribonucleases/metabolism/genetics ; Humans ; Embryoid Bodies/cytology/metabolism ; Bacterial Proteins/metabolism/genetics ; },
abstract = {Single-cell lineage tracing based on CRISPR-Cas9 gene editing enables the simultaneous linkage of cell states and lineage history at a high resolution. Despite its immense potential in resolving the cell fate determination and genealogy within an organism, existing implementations of this technology suffer from limitations in recording capabilities and considerable barcode dropout. Here, we introduce DuTracer, a versatile tool that utilizes two orthogonal gene editing systems to record cell lineage history at single-cell resolution in an inducible manner. DuTracer shows the ability to enhance lineage recording with minimized target dropouts and potentially deeper tree depths. Applying DuTracer in mouse embryoid bodies and neuromesodermal organoids illustrates the lineage relationship of different cell types and proposes potential lineage-biased molecular drivers, showcased by identifying transcription factor Foxb1 as a modulator in the cell fate determination of neuromesodermal progenitors. Collectively, DuTracer facilitates the precise and regulatory interrogation of cellular lineages of complex biological processes.},
}
MeSH Terms:
show MeSH Terms
hide MeSH Terms
Animals
*Cell Lineage
Mice
*Single-Cell Analysis/methods
*CRISPR-Cas Systems/genetics
Gene Editing/methods
CRISPR-Associated Protein 9/metabolism
CRISPR-Associated Proteins/metabolism/genetics
Cell Differentiation
Endodeoxyribonucleases/metabolism/genetics
Humans
Embryoid Bodies/cytology/metabolism
Bacterial Proteins/metabolism/genetics
RevDate: 2025-01-31
CmpDate: 2025-01-31
Compact RNA editors with natural miniature Cas13j nucleases.
Nature chemical biology, 21(2):280-290.
Clustered regularly interspaced short palindromic repeats-Cas13 effectors are used for RNA editing but the adeno-associated virus (AAV) packaging limitations because of their big sizes hinder their therapeutic application. Here we report the identification of the Cas13j family, with LepCas13j (529 aa) and ChiCas13j (424 aa) being the smallest and most highly efficient variants for RNA interference. The miniaturized Cas13j proteins enable the development of compact RNA base editors. Chi-RESCUE-S, by fusing dChiCas13j with hADAR2dd, demonstrates high efficiency and specificity in A-to-G and C-to-U conversions. Importantly, this system is compatible with single-AAV packaging without the need for protein sequence truncation. It successfully corrected pathogenic mutations, such as APOC3[D65N] and SCN9A[R896Q], to the wild-type forms. In addition, we developed an optimized system, Chi-RESCUE-S-mini3, which pioneered efficient in vivo C-to-U RNA editing of PCSK9 in mice through single-AAV delivery, resulting in reduced total cholesterol levels. These results highlight the potential of Cas13j to treat human diseases.
Additional Links: PMID-39300230
PubMed:
Citation:
show bibtex listing
hide bibtex listing
@article {pmid39300230,
year = {2025},
author = {Li, G and Cheng, Y and Yu, J and Zhu, Y and Ma, H and Zhou, Y and Pu, Z and Zhu, G and Yuan, Y and Zhang, Z and Zhou, X and Tian, K and Qiao, J and Hu, X and Chen, XX and Ji, Q and Huang, X and Ma, B and Yao, Y},
title = {Compact RNA editors with natural miniature Cas13j nucleases.},
journal = {Nature chemical biology},
volume = {21},
number = {2},
pages = {280-290},
pmid = {39300230},
issn = {1552-4469},
support = {2023YFC3402402//Ministry of Science and Technology of the People's Republic of China (Chinese Ministry of Science and Technology)/ ; 02020200-K02013008//Zhejiang University (ZJU)/ ; },
mesh = {Animals ; Mice ; *Dependovirus/genetics ; Humans ; RNA Editing ; Proprotein Convertase 9/genetics/metabolism ; HEK293 Cells ; CRISPR-Cas Systems ; CRISPR-Associated Proteins/metabolism/genetics ; RNA/genetics/metabolism ; },
abstract = {Clustered regularly interspaced short palindromic repeats-Cas13 effectors are used for RNA editing but the adeno-associated virus (AAV) packaging limitations because of their big sizes hinder their therapeutic application. Here we report the identification of the Cas13j family, with LepCas13j (529 aa) and ChiCas13j (424 aa) being the smallest and most highly efficient variants for RNA interference. The miniaturized Cas13j proteins enable the development of compact RNA base editors. Chi-RESCUE-S, by fusing dChiCas13j with hADAR2dd, demonstrates high efficiency and specificity in A-to-G and C-to-U conversions. Importantly, this system is compatible with single-AAV packaging without the need for protein sequence truncation. It successfully corrected pathogenic mutations, such as APOC3[D65N] and SCN9A[R896Q], to the wild-type forms. In addition, we developed an optimized system, Chi-RESCUE-S-mini3, which pioneered efficient in vivo C-to-U RNA editing of PCSK9 in mice through single-AAV delivery, resulting in reduced total cholesterol levels. These results highlight the potential of Cas13j to treat human diseases.},
}
MeSH Terms:
show MeSH Terms
hide MeSH Terms
Animals
Mice
*Dependovirus/genetics
Humans
RNA Editing
Proprotein Convertase 9/genetics/metabolism
HEK293 Cells
CRISPR-Cas Systems
CRISPR-Associated Proteins/metabolism/genetics
RNA/genetics/metabolism
RevDate: 2025-01-30
H9N2 avian influenza virus diagnostics utilizing specific high-sensitivity enzymatic molecular system termed RPA-based CRISPR-Cas13a.
International journal of biological macromolecules pii:S0141-8130(25)01023-2 [Epub ahead of print].
H9N2 avian influenza virus (AIV), a major pathogen causing respiratory infections in poultry, poses a significant threat to the poultry industry and human health. Early detection and control of H9N2 infections are essential for minimizing economic losses and preventing potential zoonotic transmission. A novel CRISPR-Cas family member called CRISPR-Cas13a comprises the CRISPR RNA (crRNA) and Cas13a nuclease. Through the crRNA-based reprogramming of Cas13a, a platform for sensing RNAs specifically is available. In this study, we developed a RPA-based CRISPR-Cas13a diagnostic method for rapid detection of the H9N2 AIV. The results demonstrated that at a limit of 10 copies/μL and 10[2] copies/μL could be detected within 50 min, by fluorescence detection and lateral flow strip, respectively, offering a highly sensitive method for H9N2 detection. This method exhibited excellent specificity, distinguishing H9N2 from other pathogens. Furthermore, the RPA-Cas13a-based detection system was tested on clinical samples, showing comparable performance to RT-qPCR. The detection results were visualized using either lateral flow assays or fluorescence, making it a suitable tool for on-site, field-deployable diagnostics. In a word, this RPA-Cas13a diagnostic approach offers high reliability, sensitivity, and specificity, with promising potential for rapidly detecting H9N2 and other viral pathogens in clinical and food safety applications.
Additional Links: PMID-39884612
Publisher:
PubMed:
Citation:
show bibtex listing
hide bibtex listing
@article {pmid39884612,
year = {2025},
author = {He, D and Zhao, S and Wang, F and Wu, B and Wei, F and Zhao, Y and Wei, X and Ren, H and Zhang, M and Fan, Y and Zhang, J and Yu, S and Tang, Y and Diao, Y},
title = {H9N2 avian influenza virus diagnostics utilizing specific high-sensitivity enzymatic molecular system termed RPA-based CRISPR-Cas13a.},
journal = {International journal of biological macromolecules},
volume = {},
number = {},
pages = {140474},
doi = {10.1016/j.ijbiomac.2025.140474},
pmid = {39884612},
issn = {1879-0003},
abstract = {H9N2 avian influenza virus (AIV), a major pathogen causing respiratory infections in poultry, poses a significant threat to the poultry industry and human health. Early detection and control of H9N2 infections are essential for minimizing economic losses and preventing potential zoonotic transmission. A novel CRISPR-Cas family member called CRISPR-Cas13a comprises the CRISPR RNA (crRNA) and Cas13a nuclease. Through the crRNA-based reprogramming of Cas13a, a platform for sensing RNAs specifically is available. In this study, we developed a RPA-based CRISPR-Cas13a diagnostic method for rapid detection of the H9N2 AIV. The results demonstrated that at a limit of 10 copies/μL and 10[2] copies/μL could be detected within 50 min, by fluorescence detection and lateral flow strip, respectively, offering a highly sensitive method for H9N2 detection. This method exhibited excellent specificity, distinguishing H9N2 from other pathogens. Furthermore, the RPA-Cas13a-based detection system was tested on clinical samples, showing comparable performance to RT-qPCR. The detection results were visualized using either lateral flow assays or fluorescence, making it a suitable tool for on-site, field-deployable diagnostics. In a word, this RPA-Cas13a diagnostic approach offers high reliability, sensitivity, and specificity, with promising potential for rapidly detecting H9N2 and other viral pathogens in clinical and food safety applications.},
}
RevDate: 2025-01-30
Exosome-mediated CRISPR/Cas delivery: A cutting-edge frontier in cancer gene therapy.
Gene pii:S0378-1119(25)00084-8 [Epub ahead of print].
Cancer is considered the second most common disease globally. In the past few decades, many approaches have been proposed for cancer treatment. One among those is targeted therapy using CRISPR-Cas system which plays an irreplaceable role in translational research through gene editing. However, due to its inability to cope with specific targeting, off-target effects, and limited tumor penetration, it is very challenging to use this approach in cancer studies. To increase its efficacy, CRISPR components are engineered into the extracellular vesicles (EVs), especially exosomes (a subpopulation of EVs). Exosomes have a significant role in cellular communication. Exosome-based CRISPR-Cas system transport for gene editing enhanced specificity, reduced off-target effects, and improved therapeutic potential. In this review, we highlighted the role of exosomes and the CRISPR-Cas system in cancer research, exosome-based CRISPR delivery for cancer treatment, and its future orientation.
Additional Links: PMID-39884405
Publisher:
PubMed:
Citation:
show bibtex listing
hide bibtex listing
@article {pmid39884405,
year = {2025},
author = {S, BR and Dhar, R and Devi, A},
title = {Exosome-mediated CRISPR/Cas delivery: A cutting-edge frontier in cancer gene therapy.},
journal = {Gene},
volume = {},
number = {},
pages = {149296},
doi = {10.1016/j.gene.2025.149296},
pmid = {39884405},
issn = {1879-0038},
abstract = {Cancer is considered the second most common disease globally. In the past few decades, many approaches have been proposed for cancer treatment. One among those is targeted therapy using CRISPR-Cas system which plays an irreplaceable role in translational research through gene editing. However, due to its inability to cope with specific targeting, off-target effects, and limited tumor penetration, it is very challenging to use this approach in cancer studies. To increase its efficacy, CRISPR components are engineered into the extracellular vesicles (EVs), especially exosomes (a subpopulation of EVs). Exosomes have a significant role in cellular communication. Exosome-based CRISPR-Cas system transport for gene editing enhanced specificity, reduced off-target effects, and improved therapeutic potential. In this review, we highlighted the role of exosomes and the CRISPR-Cas system in cancer research, exosome-based CRISPR delivery for cancer treatment, and its future orientation.},
}
RevDate: 2025-01-30
CmpDate: 2025-01-30
Randomizing the human genome by engineering recombination between repeat elements.
Science (New York, N.Y.), 387(6733):eado3979.
We lack tools to edit DNA sequences at scales necessary to study 99% of the human genome that is noncoding. To address this gap, we applied CRISPR prime editing to insert recombination handles into repetitive sequences, up to 1697 per cell line, which enables generating large-scale deletions, inversions, translocations, and circular DNA. Recombinase induction produced more than 100 stochastic megabase-sized rearrangements in each cell. We tracked these rearrangements over time to measure selection pressures, finding a preference for shorter variants that avoided essential genes. We characterized 29 clones with multiple rearrangements, finding an impact of deletions on expression of genes in the variant but not on nearby genes. This genome-scrambling strategy enables large deletions, sequence relocations, and the insertion of regulatory elements to explore genome dispensability and organization.
Additional Links: PMID-39883775
Publisher:
PubMed:
Citation:
show bibtex listing
hide bibtex listing
@article {pmid39883775,
year = {2025},
author = {Koeppel, J and Ferreira, R and Vanderstichele, T and Riedmayr, LM and Peets, EM and Girling, G and Weller, J and Murat, P and Liberante, FG and Ellis, T and Church, GM and Parts, L},
title = {Randomizing the human genome by engineering recombination between repeat elements.},
journal = {Science (New York, N.Y.)},
volume = {387},
number = {6733},
pages = {eado3979},
doi = {10.1126/science.ado3979},
pmid = {39883775},
issn = {1095-9203},
mesh = {Humans ; *Genome, Human ; *Repetitive Sequences, Nucleic Acid ; *Gene Editing/methods ; *CRISPR-Cas Systems ; *Recombination, Genetic ; Translocation, Genetic ; Sequence Deletion ; Gene Rearrangement ; Chromosome Inversion ; Genes, Essential ; Genetic Engineering/methods ; },
abstract = {We lack tools to edit DNA sequences at scales necessary to study 99% of the human genome that is noncoding. To address this gap, we applied CRISPR prime editing to insert recombination handles into repetitive sequences, up to 1697 per cell line, which enables generating large-scale deletions, inversions, translocations, and circular DNA. Recombinase induction produced more than 100 stochastic megabase-sized rearrangements in each cell. We tracked these rearrangements over time to measure selection pressures, finding a preference for shorter variants that avoided essential genes. We characterized 29 clones with multiple rearrangements, finding an impact of deletions on expression of genes in the variant but not on nearby genes. This genome-scrambling strategy enables large deletions, sequence relocations, and the insertion of regulatory elements to explore genome dispensability and organization.},
}
MeSH Terms:
show MeSH Terms
hide MeSH Terms
Humans
*Genome, Human
*Repetitive Sequences, Nucleic Acid
*Gene Editing/methods
*CRISPR-Cas Systems
*Recombination, Genetic
Translocation, Genetic
Sequence Deletion
Gene Rearrangement
Chromosome Inversion
Genes, Essential
Genetic Engineering/methods
RevDate: 2025-01-30
CmpDate: 2025-01-30
Direct repeat region 3' end modifications regulate Cas12a activity and expand its applications.
Nucleic acids research, 53(3):.
CRISPR-Cas12a technology has transformative potential, but as its applications grow, enhancing its inherent functionalities is essential to meet diverse demands. Here, we reveal a regulatory mechanism for LbCas12a through direct repeat (DR) region 3' end modifications and de-modifications, which can regulate LbCas12a's cis- and trans-cleavage activities. We extensively explored the effects of introducing phosphorylation, DNA, photo-cleavable linker, DNA modifications at the DR 3' end on LbCas12a's functionality. We find that the temporary inhibitory function of Cas12a can be reactivated by DR 3' end modification corresponding substances, such as alkaline phosphatase (ALP), immunoglobulin G (IgG), alpha-fetoprotein (AFP), DNA exonucleases, ultraviolet radiation, and DNA glycosylases, which greatly expand the scope of application of Cas12a. Clinical applications demonstrated promising results in ALP, AFP, and trace Epstein-Barr virus detection compared to gold standard methods. Our research provides valuable insights into regulating LbCas12a activity through direct modification of DR and significantly expands its potential clinical detection targets, paving the way for future universal clustered regularly interspaced short palindromic repeats (CRISPR) diagnostic strategies.
Additional Links: PMID-39883010
Publisher:
PubMed:
Citation:
show bibtex listing
hide bibtex listing
@article {pmid39883010,
year = {2025},
author = {Zhang, W and Zhong, Y and Wang, J and Zou, G and Chen, Q and Liu, C},
title = {Direct repeat region 3' end modifications regulate Cas12a activity and expand its applications.},
journal = {Nucleic acids research},
volume = {53},
number = {3},
pages = {},
doi = {10.1093/nar/gkaf040},
pmid = {39883010},
issn = {1362-4962},
support = {22307150//National Natural Science Foundation of China/ ; A2303012//Shenzhen Medical Research Fund/ ; 2024A1515012319//Guangdong Basic and Applied Basic Research Foundation/ ; JCYJ20230807110315032//Shenzhen Science and Technology Program/ ; },
mesh = {*CRISPR-Cas Systems ; *CRISPR-Associated Proteins/metabolism/genetics ; Humans ; Alkaline Phosphatase/metabolism/genetics ; alpha-Fetoproteins/genetics/metabolism ; Endodeoxyribonucleases/metabolism/genetics ; Bacterial Proteins/metabolism/genetics ; Phosphorylation ; Immunoglobulin G ; DNA/metabolism/genetics/chemistry ; Herpesvirus 4, Human/genetics ; },
abstract = {CRISPR-Cas12a technology has transformative potential, but as its applications grow, enhancing its inherent functionalities is essential to meet diverse demands. Here, we reveal a regulatory mechanism for LbCas12a through direct repeat (DR) region 3' end modifications and de-modifications, which can regulate LbCas12a's cis- and trans-cleavage activities. We extensively explored the effects of introducing phosphorylation, DNA, photo-cleavable linker, DNA modifications at the DR 3' end on LbCas12a's functionality. We find that the temporary inhibitory function of Cas12a can be reactivated by DR 3' end modification corresponding substances, such as alkaline phosphatase (ALP), immunoglobulin G (IgG), alpha-fetoprotein (AFP), DNA exonucleases, ultraviolet radiation, and DNA glycosylases, which greatly expand the scope of application of Cas12a. Clinical applications demonstrated promising results in ALP, AFP, and trace Epstein-Barr virus detection compared to gold standard methods. Our research provides valuable insights into regulating LbCas12a activity through direct modification of DR and significantly expands its potential clinical detection targets, paving the way for future universal clustered regularly interspaced short palindromic repeats (CRISPR) diagnostic strategies.},
}
MeSH Terms:
show MeSH Terms
hide MeSH Terms
*CRISPR-Cas Systems
*CRISPR-Associated Proteins/metabolism/genetics
Humans
Alkaline Phosphatase/metabolism/genetics
alpha-Fetoproteins/genetics/metabolism
Endodeoxyribonucleases/metabolism/genetics
Bacterial Proteins/metabolism/genetics
Phosphorylation
Immunoglobulin G
DNA/metabolism/genetics/chemistry
Herpesvirus 4, Human/genetics
RevDate: 2025-01-29
CmpDate: 2025-01-29
Non-additive dosage-dependent effects of TaGS3 gene editing on grain size and weight in wheat.
TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik, 138(2):38.
Loss-of-function mutations induced by CRISPR-Cas9 in the TaGS3 gene homoeologs show non-additive dosage-dependent effects on grain size and weight and have potential utility for increasing grain yield in wheat. The grain size in cereals is one of the component traits contributing to yield. Previous studies showed that loss-of-function (LOF) mutations in GS3, encoding Gγ subunit of the multimeric G protein complex, increase grain size and weight in rice. While an association between allelic variation in the GS3 homologs of wheat and grain weight/size has been detected previously, the effects of LOF alleles at TaGS3 on these traits remain unknown. We used genome editing to create TaGS3 mutant lines with varying LOF homeo-allele dosages. Contrary to the results obtained in rice, editing all three TaGS3 homoeologous copies resulted in a significant decrease in grain length (4.4%), width (3.4%), grain area (7.3%) and weight (7.5%), without affecting the number of grains per spike. Compared to the wild type, the highest increase in grain weight (up to 9.6%) and area (up to 5.0%) was observed in homozygous mutants with one or two genomes carrying LOF homeo-alleles, suggesting non-additive suppressive effects of TaGS3 on grain size and weight in wheat. Our results suggest that the regulatory effects of GS3 homologs in wheat and rice have diverged. The newly developed LOF homeo-alleles of TaGS3 expand the set of CRISPR-Cas9-induced variants of yield component genes that have potential to increase grain weight in wheat.
Additional Links: PMID-39880939
PubMed:
Citation:
show bibtex listing
hide bibtex listing
@article {pmid39880939,
year = {2025},
author = {Wang, W and Pan, Q and Tian, B and Yu, Z and Davidson, D and Bai, G and Akhunova, A and Trick, H and Akhunov, E},
title = {Non-additive dosage-dependent effects of TaGS3 gene editing on grain size and weight in wheat.},
journal = {TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik},
volume = {138},
number = {2},
pages = {38},
pmid = {39880939},
issn = {1432-2242},
support = {2021-67013-34174//National Institute of Food and Agriculture/ ; 2020-67013-30906//National Institute of Food and Agriculture/ ; 2022-68013-36439//National Institute of Food and Agriculture/ ; INV-004430/GATES/Bill & Melinda Gates Foundation/United States ; },
mesh = {*Triticum/genetics/growth & development ; *Gene Editing/methods ; *Edible Grain/genetics/growth & development ; *CRISPR-Cas Systems ; Alleles ; Phenotype ; Plant Proteins/genetics ; Seeds/growth & development/genetics ; Gene Dosage ; Genes, Plant ; Plants, Genetically Modified/growth & development/genetics ; Loss of Function Mutation ; },
abstract = {Loss-of-function mutations induced by CRISPR-Cas9 in the TaGS3 gene homoeologs show non-additive dosage-dependent effects on grain size and weight and have potential utility for increasing grain yield in wheat. The grain size in cereals is one of the component traits contributing to yield. Previous studies showed that loss-of-function (LOF) mutations in GS3, encoding Gγ subunit of the multimeric G protein complex, increase grain size and weight in rice. While an association between allelic variation in the GS3 homologs of wheat and grain weight/size has been detected previously, the effects of LOF alleles at TaGS3 on these traits remain unknown. We used genome editing to create TaGS3 mutant lines with varying LOF homeo-allele dosages. Contrary to the results obtained in rice, editing all three TaGS3 homoeologous copies resulted in a significant decrease in grain length (4.4%), width (3.4%), grain area (7.3%) and weight (7.5%), without affecting the number of grains per spike. Compared to the wild type, the highest increase in grain weight (up to 9.6%) and area (up to 5.0%) was observed in homozygous mutants with one or two genomes carrying LOF homeo-alleles, suggesting non-additive suppressive effects of TaGS3 on grain size and weight in wheat. Our results suggest that the regulatory effects of GS3 homologs in wheat and rice have diverged. The newly developed LOF homeo-alleles of TaGS3 expand the set of CRISPR-Cas9-induced variants of yield component genes that have potential to increase grain weight in wheat.},
}
MeSH Terms:
show MeSH Terms
hide MeSH Terms
*Triticum/genetics/growth & development
*Gene Editing/methods
*Edible Grain/genetics/growth & development
*CRISPR-Cas Systems
Alleles
Phenotype
Plant Proteins/genetics
Seeds/growth & development/genetics
Gene Dosage
Genes, Plant
Plants, Genetically Modified/growth & development/genetics
Loss of Function Mutation
RevDate: 2025-01-29
Building the Future of Clinical Diagnostics: An Analysis of Potential Benefits and Current Barriers in CRISPR/Cas Diagnostics.
ACS synthetic biology [Epub ahead of print].
Advancements in molecular diagnostics, such as polymerase chain reaction and next-generation sequencing, have revolutionized disease management and prognosis. Despite these advancements in molecular diagnostics, the field faces challenges due to high operational costs and the need for sophisticated equipment and highly trained personnel besides having several technical limitations. The emergent field of CRISPR/Cas sensing technology is showing promise as a new paradigm in clinical diagnostics, although widespread clinical adoption remains limited. This perspective paper discusses specific cases where CRISPR/Cas technology can surmount the challenges of existing diagnostic methods by stressing the significant role that CRISPR/Cas technology can play in revolutionizing clinical diagnostics. It underscores the urgency and importance of addressing the technological and regulatory hurdles that must be overcome to harness this technology effectively in clinical laboratories.
Additional Links: PMID-39880685
Publisher:
PubMed:
Citation:
show bibtex listing
hide bibtex listing
@article {pmid39880685,
year = {2025},
author = {van Dongen, JE and Segerink, LI},
title = {Building the Future of Clinical Diagnostics: An Analysis of Potential Benefits and Current Barriers in CRISPR/Cas Diagnostics.},
journal = {ACS synthetic biology},
volume = {},
number = {},
pages = {},
doi = {10.1021/acssynbio.4c00816},
pmid = {39880685},
issn = {2161-5063},
abstract = {Advancements in molecular diagnostics, such as polymerase chain reaction and next-generation sequencing, have revolutionized disease management and prognosis. Despite these advancements in molecular diagnostics, the field faces challenges due to high operational costs and the need for sophisticated equipment and highly trained personnel besides having several technical limitations. The emergent field of CRISPR/Cas sensing technology is showing promise as a new paradigm in clinical diagnostics, although widespread clinical adoption remains limited. This perspective paper discusses specific cases where CRISPR/Cas technology can surmount the challenges of existing diagnostic methods by stressing the significant role that CRISPR/Cas technology can play in revolutionizing clinical diagnostics. It underscores the urgency and importance of addressing the technological and regulatory hurdles that must be overcome to harness this technology effectively in clinical laboratories.},
}
RevDate: 2025-01-30
CmpDate: 2025-01-30
Unlocking crops' genetic potential: Advances in genome and epigenome editing of regulatory regions.
Current opinion in plant biology, 83:102669.
Genome editing tools could precisely and efficiently target plant genomes leading to the development of improved crops. Besides editing the coding regions, researchers can employ editing technologies to target specific gene regulatory elements or modify epigenetic marks associated with distal regulatory regions, thereby regulating gene expression in crops. This review outlines several prominent genome editing technologies, including CRISPR-Cas9, TALENs, and ZFNs and recent advancements. The applications for genome and epigenome editing especially of regulatory regions in crop plants is also discussed, including efforts to enhance abiotic stress tolerance, yield, disease resistance and plant phenotype. Additionally, the review addresses the potential of epigenetic modifications, such as DNA methylation and histone modifications, to alter gene expression for crop improvement. Finally, the limitations and future scope of utilizing various genome editing tools to manipulate regulatory elements for gene regulation to unlock the full potential of these tools in plant breeding has been discussed.
Additional Links: PMID-39603170
Publisher:
PubMed:
Citation:
show bibtex listing
hide bibtex listing
@article {pmid39603170,
year = {2025},
author = {Ali, N and Singh, S and Garg, R},
title = {Unlocking crops' genetic potential: Advances in genome and epigenome editing of regulatory regions.},
journal = {Current opinion in plant biology},
volume = {83},
number = {},
pages = {102669},
doi = {10.1016/j.pbi.2024.102669},
pmid = {39603170},
issn = {1879-0356},
mesh = {*Crops, Agricultural/genetics ; *Gene Editing/methods ; *Genome, Plant/genetics ; Epigenesis, Genetic ; Epigenome/genetics ; CRISPR-Cas Systems ; Plant Breeding/methods ; },
abstract = {Genome editing tools could precisely and efficiently target plant genomes leading to the development of improved crops. Besides editing the coding regions, researchers can employ editing technologies to target specific gene regulatory elements or modify epigenetic marks associated with distal regulatory regions, thereby regulating gene expression in crops. This review outlines several prominent genome editing technologies, including CRISPR-Cas9, TALENs, and ZFNs and recent advancements. The applications for genome and epigenome editing especially of regulatory regions in crop plants is also discussed, including efforts to enhance abiotic stress tolerance, yield, disease resistance and plant phenotype. Additionally, the review addresses the potential of epigenetic modifications, such as DNA methylation and histone modifications, to alter gene expression for crop improvement. Finally, the limitations and future scope of utilizing various genome editing tools to manipulate regulatory elements for gene regulation to unlock the full potential of these tools in plant breeding has been discussed.},
}
MeSH Terms:
show MeSH Terms
hide MeSH Terms
*Crops, Agricultural/genetics
*Gene Editing/methods
*Genome, Plant/genetics
Epigenesis, Genetic
Epigenome/genetics
CRISPR-Cas Systems
Plant Breeding/methods
RevDate: 2025-01-29
CmpDate: 2025-01-29
Multiple gRNAs-assisted CRISPR/Cas12a-based portable aptasensor enabling glucometer readout for amplification-free and quantitative detection of malathion.
Analytica chimica acta, 1341:343662.
BACKGROUND: The threat of toxic malathion residues to human health has always been a serious food safety issue. The CRISPR/Cas system represents an innovative detection technology for pesticide residues, but its application to malathion detection has not been reported yet. In addition, the multiple-guide RNA (gRNA) powered-CRISPR/Cas biosensor has the advantages of being fast, sensitive and does not require pre-amplification. However, the reported multiple-gRNA CRISPR/Cas-based biosensors are largely only used for the detection of nucleic acid targets, and there are still certain challenges in detecting non-nucleic acid targets.
RESULTS: In this work, a multiplex-gRNA-assisted CRISPR/Cas12a-based portable aptasensor (MgCPA) is developed for amplification-free and quantitative detection of malathion using a glucometer. When target malathion is present in the MgCPA strategy, it specifically binds with aptamer and then activates the trans-cleavage activity of the multiplex-gRNA CRISPR/Cas12a. The activated multiple Cas12a/gRNA complexes cut invertase-HP probes on the electrode surface to obtain glucose signals with glucometer assistance. Under optimal conditions, the developed MgCPA strategy achieves satisfactory portable quantitative and sensitive detection of malathion down to 300 fM (S/N = 3) without pre-amplification. Moreover, the satisfactory selectivity, high reproducibility, and good stability of the proposed strategy are also obtained. Due to its excellent and robust shelf life, our developed MgCPA strategy can be practically applied in detecting malathion in orange, apple, cabbage, and spinach samples.
SIGNIFICANCE: Amplification-free, sensitive, portable quantitative and selective detection of malathion in food samples is achieved by employing our developed MgCPA strategy. This strategy not only opens up a new path for the non-nucleic-acid target detection using amplification-free methods based on multiple-gRNA-assisted CRISPR/Cas12a, but also has broad application prospects in ensuring food safety.
Additional Links: PMID-39880501
Publisher:
PubMed:
Citation:
show bibtex listing
hide bibtex listing
@article {pmid39880501,
year = {2025},
author = {Tian, Y and Chen, J and Chen, F and Xu, J and Huang, L and Peng, L and Li, H and Shi, K},
title = {Multiple gRNAs-assisted CRISPR/Cas12a-based portable aptasensor enabling glucometer readout for amplification-free and quantitative detection of malathion.},
journal = {Analytica chimica acta},
volume = {1341},
number = {},
pages = {343662},
doi = {10.1016/j.aca.2025.343662},
pmid = {39880501},
issn = {1873-4324},
mesh = {*Malathion/analysis ; *CRISPR-Cas Systems ; *Biosensing Techniques ; *Aptamers, Nucleotide/chemistry ; *RNA, Guide, CRISPR-Cas Systems ; Limit of Detection ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {BACKGROUND: The threat of toxic malathion residues to human health has always been a serious food safety issue. The CRISPR/Cas system represents an innovative detection technology for pesticide residues, but its application to malathion detection has not been reported yet. In addition, the multiple-guide RNA (gRNA) powered-CRISPR/Cas biosensor has the advantages of being fast, sensitive and does not require pre-amplification. However, the reported multiple-gRNA CRISPR/Cas-based biosensors are largely only used for the detection of nucleic acid targets, and there are still certain challenges in detecting non-nucleic acid targets.
RESULTS: In this work, a multiplex-gRNA-assisted CRISPR/Cas12a-based portable aptasensor (MgCPA) is developed for amplification-free and quantitative detection of malathion using a glucometer. When target malathion is present in the MgCPA strategy, it specifically binds with aptamer and then activates the trans-cleavage activity of the multiplex-gRNA CRISPR/Cas12a. The activated multiple Cas12a/gRNA complexes cut invertase-HP probes on the electrode surface to obtain glucose signals with glucometer assistance. Under optimal conditions, the developed MgCPA strategy achieves satisfactory portable quantitative and sensitive detection of malathion down to 300 fM (S/N = 3) without pre-amplification. Moreover, the satisfactory selectivity, high reproducibility, and good stability of the proposed strategy are also obtained. Due to its excellent and robust shelf life, our developed MgCPA strategy can be practically applied in detecting malathion in orange, apple, cabbage, and spinach samples.
SIGNIFICANCE: Amplification-free, sensitive, portable quantitative and selective detection of malathion in food samples is achieved by employing our developed MgCPA strategy. This strategy not only opens up a new path for the non-nucleic-acid target detection using amplification-free methods based on multiple-gRNA-assisted CRISPR/Cas12a, but also has broad application prospects in ensuring food safety.},
}
MeSH Terms:
show MeSH Terms
hide MeSH Terms
*Malathion/analysis
*CRISPR-Cas Systems
*Biosensing Techniques
*Aptamers, Nucleotide/chemistry
*RNA, Guide, CRISPR-Cas Systems
Limit of Detection
Bacterial Proteins
Endodeoxyribonucleases
CRISPR-Associated Proteins
RevDate: 2025-01-29
CmpDate: 2025-01-29
Application and development of CRISPR-Cas12a methods for the molecular diagnosis of cancer: A review.
Analytica chimica acta, 1341:343603.
Rapid, sensitive, and specific molecular detection methods are crucial for diagnosing, treating and prognosing cancer patients. With advancements in biotechnology, molecular diagnostic technology has garnered significant attention as a fast and accurate method for cancer diagnosis. CRISPR-Cas12a (Cpf1), an important CRISPR-Cas family member, has revolutionized the field of molecular diagnosis since its introduction. CRISPR-Cas technologies are a new generation of molecular tools that are widely used in the detection of pathogens, cancers, and other diseases. Liquid biopsy methods based on CRISPR-Cas12a have demonstrated remarkable success in cancer diagnosis, encompassing the detection of DNA mutations, DNA methylation, tumor-related viruses, and non-nucleic acid molecule identification. This review systematically discusses the developmental history, key technologies, and principles of CRISPR-Cas12a-based molecular diagnostic techniques and their applications in cancer diagnosis. This review has also discussed the future development directions of CRISPR-Cas12a, aiming for it to become a reliable new technology that can be used in clinical application.
Additional Links: PMID-39880493
Publisher:
PubMed:
Citation:
show bibtex listing
hide bibtex listing
@article {pmid39880493,
year = {2025},
author = {Wang, S and Shen, X and Chen, G and Zhang, W and Tan, B},
title = {Application and development of CRISPR-Cas12a methods for the molecular diagnosis of cancer: A review.},
journal = {Analytica chimica acta},
volume = {1341},
number = {},
pages = {343603},
doi = {10.1016/j.aca.2024.343603},
pmid = {39880493},
issn = {1873-4324},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Neoplasms/diagnosis/genetics ; Molecular Diagnostic Techniques/methods ; },
abstract = {Rapid, sensitive, and specific molecular detection methods are crucial for diagnosing, treating and prognosing cancer patients. With advancements in biotechnology, molecular diagnostic technology has garnered significant attention as a fast and accurate method for cancer diagnosis. CRISPR-Cas12a (Cpf1), an important CRISPR-Cas family member, has revolutionized the field of molecular diagnosis since its introduction. CRISPR-Cas technologies are a new generation of molecular tools that are widely used in the detection of pathogens, cancers, and other diseases. Liquid biopsy methods based on CRISPR-Cas12a have demonstrated remarkable success in cancer diagnosis, encompassing the detection of DNA mutations, DNA methylation, tumor-related viruses, and non-nucleic acid molecule identification. This review systematically discusses the developmental history, key technologies, and principles of CRISPR-Cas12a-based molecular diagnostic techniques and their applications in cancer diagnosis. This review has also discussed the future development directions of CRISPR-Cas12a, aiming for it to become a reliable new technology that can be used in clinical application.},
}
MeSH Terms:
show MeSH Terms
hide MeSH Terms
Humans
*CRISPR-Cas Systems/genetics
*Neoplasms/diagnosis/genetics
Molecular Diagnostic Techniques/methods
RevDate: 2025-01-29
Cas12a Is Competitive for Gene Editing in the Malaria parasites.
Microbial pathogenesis pii:S0882-4010(25)00065-8 [Epub ahead of print].
Malaria, caused by the Plasmodium parasites, has always been one of the worst infectious diseases that threaten human health, making it necessary for us to study the genetic function and physiological mechanisms of Plasmodium parasites from the molecular level to find more effective ways of addressing the increasingly pressing threat. The CRISPR (Clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated protein) is an RNA-guided adaptive immune system, which has been extensively developed and used as a genome editing tool in many organisms, including Plasmodium parasites. However, due to the physiological characteristics and special genomic characteristics of Plasmodium parasites, most of the tools currently used for genome editing of Plasmodium parasites have not met expectations. CRISPR-Cas12a (also known as Cpf1), one of the CRISPR-Cas systems, has attracted considerable attention because of its characteristics of being used for biological diagnosis and multiple genome editing. Recent studies have shown that its unique properties fit the genetic makeup of Plasmodium parasites making it a promising tool for gene editing in these parasites. In this review, we have summarized the relevant content of the Cas12 family, especially the frequently used Cas12a, its advantages for gene editing, and the application prospects in Plasmodium parasites.
Additional Links: PMID-39880137
Publisher:
PubMed:
Citation:
show bibtex listing
hide bibtex listing
@article {pmid39880137,
year = {2025},
author = {Yang, S and Wei, Y and Quansah, E and Zhang, Z and Da, W and Wang, B and Wang, K and Sun, D and Tao, Z and Zhang, C},
title = {Cas12a Is Competitive for Gene Editing in the Malaria parasites.},
journal = {Microbial pathogenesis},
volume = {},
number = {},
pages = {107340},
doi = {10.1016/j.micpath.2025.107340},
pmid = {39880137},
issn = {1096-1208},
abstract = {Malaria, caused by the Plasmodium parasites, has always been one of the worst infectious diseases that threaten human health, making it necessary for us to study the genetic function and physiological mechanisms of Plasmodium parasites from the molecular level to find more effective ways of addressing the increasingly pressing threat. The CRISPR (Clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated protein) is an RNA-guided adaptive immune system, which has been extensively developed and used as a genome editing tool in many organisms, including Plasmodium parasites. However, due to the physiological characteristics and special genomic characteristics of Plasmodium parasites, most of the tools currently used for genome editing of Plasmodium parasites have not met expectations. CRISPR-Cas12a (also known as Cpf1), one of the CRISPR-Cas systems, has attracted considerable attention because of its characteristics of being used for biological diagnosis and multiple genome editing. Recent studies have shown that its unique properties fit the genetic makeup of Plasmodium parasites making it a promising tool for gene editing in these parasites. In this review, we have summarized the relevant content of the Cas12 family, especially the frequently used Cas12a, its advantages for gene editing, and the application prospects in Plasmodium parasites.},
}
RevDate: 2025-01-29
CmpDate: 2025-01-29
Targeted insertion of heterogenous DNA using Cas9-gRNA ribonucleoprotein-mediated gene editing in Ganoderma lucidum.
Bioengineered, 16(1):2458376.
Gene editing is emerging as a powerful tool for introducing novel functionalities in mushrooms. While CRISPR/Cas9-induced double-strand breaks (DSBs) typically rely on non-homologous end joining (NHEJ) for gene disruption, precise insertion of heterologous DNA in mushrooms is less explored. Here, we evaluated the efficacy of inserting donor DNAs (8-1008 bp) with or without homologous arms at Cas9-gRNA RNP-induced DSBs. Co-transformation of donor DNAs with RNP targeting the pyrG gene in Ganoderma lucidum yielded 184 transformants without homologous arms and 781 with 300-bp homologous arms (HR_donor DNAs). Restriction analysis and sequencing identified 122 hR_donor DNA transformants with complete donor DNA sequences, achieving 15.6% HDR efficiency (122/781), contrasting with 8 instances via NHEJ from the 184 transformants. These findings highlight the viability of HDR for precise genomic editing in mushrooms, enabling targeted modifications to enhance functionalities.
Additional Links: PMID-39879084
Publisher:
PubMed:
Citation:
show bibtex listing
hide bibtex listing
@article {pmid39879084,
year = {2025},
author = {Eom, H and Choi, YJ and Nandre, R and Kim, M and Oh, YL and Kim, S and Nakazawa, T and Honda, Y and Ro, HS},
title = {Targeted insertion of heterogenous DNA using Cas9-gRNA ribonucleoprotein-mediated gene editing in Ganoderma lucidum.},
journal = {Bioengineered},
volume = {16},
number = {1},
pages = {2458376},
doi = {10.1080/21655979.2025.2458376},
pmid = {39879084},
issn = {2165-5987},
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; *Reishi/genetics/metabolism ; *RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; *Ribonucleoproteins/genetics/metabolism ; CRISPR-Associated Protein 9/genetics/metabolism ; DNA Breaks, Double-Stranded ; DNA, Fungal/genetics/metabolism ; },
abstract = {Gene editing is emerging as a powerful tool for introducing novel functionalities in mushrooms. While CRISPR/Cas9-induced double-strand breaks (DSBs) typically rely on non-homologous end joining (NHEJ) for gene disruption, precise insertion of heterologous DNA in mushrooms is less explored. Here, we evaluated the efficacy of inserting donor DNAs (8-1008 bp) with or without homologous arms at Cas9-gRNA RNP-induced DSBs. Co-transformation of donor DNAs with RNP targeting the pyrG gene in Ganoderma lucidum yielded 184 transformants without homologous arms and 781 with 300-bp homologous arms (HR_donor DNAs). Restriction analysis and sequencing identified 122 hR_donor DNA transformants with complete donor DNA sequences, achieving 15.6% HDR efficiency (122/781), contrasting with 8 instances via NHEJ from the 184 transformants. These findings highlight the viability of HDR for precise genomic editing in mushrooms, enabling targeted modifications to enhance functionalities.},
}
MeSH Terms:
show MeSH Terms
hide MeSH Terms
*Gene Editing/methods
*CRISPR-Cas Systems/genetics
*Reishi/genetics/metabolism
*RNA, Guide, CRISPR-Cas Systems/genetics/metabolism
*Ribonucleoproteins/genetics/metabolism
CRISPR-Associated Protein 9/genetics/metabolism
DNA Breaks, Double-Stranded
DNA, Fungal/genetics/metabolism
RevDate: 2025-01-29
CmpDate: 2025-01-29
Deletion of atypical type II restriction genes in Clostridium cellulovorans using a Cas9-based gene editing system.
Applied microbiology and biotechnology, 109(1):31.
The anaerobic bacterium Clostridium cellulovorans is a promising candidate for the sustainable production of biofuels and platform chemicals due to its cellulolytic properties. However, the genomic engineering of the species is hampered because of its poor genetic accessibility and the lack of genetic tools. To overcome this limitation, a protocol for triparental conjugation was established that enables the reliable transfer of vectors for markerless chromosomal modification into C. cellulovorans. The availability of reporter genes is another requirement for strain engineering and biotechnological applications. In this work, the oxygen-free fluorescence absorption-shift tag (FAST) system was used to characterize promoter strength in C. cellulovorans. Selected promoters were used to establish a CRISPR/Cas system for markerless chromosomal modifications. For stringent control of expression of Cas9, a theophylline-dependent riboswitch was used, and additionally, the anti-CRISPR protein AcrIIA4 was used to reduce the basal activity of the Cas9 in the off-state of the riboswitch. Finally, the newly established CRISPR/Cas system was used for the markerless deletion of the genes encoding two restriction endonucleases of a type II restriction-modification (RS) system from the chromosome of C. cellulovorans. In comparison to the WT, the conjugation efficiency when using the deletion mutant as the recipient strain was improved by about one order of magnitude, without the need for prior C. cellulovorans-specific in vivo methylation of the conjugative plasmid in the E. coli donor strain. KEY POINTS: • Quantification of heterologous promoters enables rational choice for genetic engineering. • CRISPR/Cas with riboswitch and anti-CRISPR allows efficient gene deletion in C. cellulovorans. • Conjugation protocol and type II REase deletion enhance genetic accessibility.
Additional Links: PMID-39878871
PubMed:
Citation:
show bibtex listing
hide bibtex listing
@article {pmid39878871,
year = {2025},
author = {Schöllkopf, AI and Almeida, L and Krammer, K and Rivero, CG and Liebl, W and Ehrenreich, A},
title = {Deletion of atypical type II restriction genes in Clostridium cellulovorans using a Cas9-based gene editing system.},
journal = {Applied microbiology and biotechnology},
volume = {109},
number = {1},
pages = {31},
pmid = {39878871},
issn = {1432-0614},
support = {161B0930//Bundesministerium für Bildung und Forschung/ ; },
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems ; *Clostridium cellulovorans/genetics/metabolism ; Promoter Regions, Genetic ; Gene Deletion ; CRISPR-Associated Protein 9/genetics/metabolism ; Riboswitch/genetics ; Conjugation, Genetic ; Bacterial Proteins/genetics/metabolism ; },
abstract = {The anaerobic bacterium Clostridium cellulovorans is a promising candidate for the sustainable production of biofuels and platform chemicals due to its cellulolytic properties. However, the genomic engineering of the species is hampered because of its poor genetic accessibility and the lack of genetic tools. To overcome this limitation, a protocol for triparental conjugation was established that enables the reliable transfer of vectors for markerless chromosomal modification into C. cellulovorans. The availability of reporter genes is another requirement for strain engineering and biotechnological applications. In this work, the oxygen-free fluorescence absorption-shift tag (FAST) system was used to characterize promoter strength in C. cellulovorans. Selected promoters were used to establish a CRISPR/Cas system for markerless chromosomal modifications. For stringent control of expression of Cas9, a theophylline-dependent riboswitch was used, and additionally, the anti-CRISPR protein AcrIIA4 was used to reduce the basal activity of the Cas9 in the off-state of the riboswitch. Finally, the newly established CRISPR/Cas system was used for the markerless deletion of the genes encoding two restriction endonucleases of a type II restriction-modification (RS) system from the chromosome of C. cellulovorans. In comparison to the WT, the conjugation efficiency when using the deletion mutant as the recipient strain was improved by about one order of magnitude, without the need for prior C. cellulovorans-specific in vivo methylation of the conjugative plasmid in the E. coli donor strain. KEY POINTS: • Quantification of heterologous promoters enables rational choice for genetic engineering. • CRISPR/Cas with riboswitch and anti-CRISPR allows efficient gene deletion in C. cellulovorans. • Conjugation protocol and type II REase deletion enhance genetic accessibility.},
}
MeSH Terms:
show MeSH Terms
hide MeSH Terms
*Gene Editing/methods
*CRISPR-Cas Systems
*Clostridium cellulovorans/genetics/metabolism
Promoter Regions, Genetic
Gene Deletion
CRISPR-Associated Protein 9/genetics/metabolism
Riboswitch/genetics
Conjugation, Genetic
Bacterial Proteins/genetics/metabolism
▼ ▼ LOAD NEXT 100 CITATIONS
ESP Quick Facts
ESP Origins
In the early 1990's, Robert Robbins was a faculty member at Johns Hopkins, where he directed the informatics core of GDB — the human gene-mapping database of the international human genome project. To share papers with colleagues around the world, he set up a small paper-sharing section on his personal web page. This small project evolved into The Electronic Scholarly Publishing Project.
ESP Support
In 1995, Robbins became the VP/IT of the Fred Hutchinson Cancer Research Center in Seattle, WA. Soon after arriving in Seattle, Robbins secured funding, through the ELSI component of the US Human Genome Project, to create the original ESP.ORG web site, with the formal goal of providing free, world-wide access to the literature of classical genetics.
ESP Rationale
Although the methods of molecular biology can seem almost magical to the uninitiated, the original techniques of classical genetics are readily appreciated by one and all: cross individuals that differ in some inherited trait, collect all of the progeny, score their attributes, and propose mechanisms to explain the patterns of inheritance observed.
ESP Goal
In reading the early works of classical genetics, one is drawn, almost inexorably, into ever more complex models, until molecular explanations begin to seem both necessary and natural. At that point, the tools for understanding genome research are at hand. Assisting readers reach this point was the original goal of The Electronic Scholarly Publishing Project.
ESP Usage
Usage of the site grew rapidly and has remained high. Faculty began to use the site for their assigned readings. Other on-line publishers, ranging from The New York Times to Nature referenced ESP materials in their own publications. Nobel laureates (e.g., Joshua Lederberg) regularly used the site and even wrote to suggest changes and improvements.
ESP Content
When the site began, no journals were making their early content available in digital format. As a result, ESP was obliged to digitize classic literature before it could be made available. For many important papers — such as Mendel's original paper or the first genetic map — ESP had to produce entirely new typeset versions of the works, if they were to be available in a high-quality format.
ESP Help
Early support from the DOE component of the Human Genome Project was critically important for getting the ESP project on a firm foundation. Since that funding ended (nearly 20 years ago), the project has been operated as a purely volunteer effort. Anyone wishing to assist in these efforts should send an email to Robbins.
ESP Plans
With the development of methods for adding typeset side notes to PDF files, the ESP project now plans to add annotated versions of some classical papers to its holdings. We also plan to add new reference and pedagogical material. We have already started providing regularly updated, comprehensive bibliographies to the ESP.ORG site.
ESP Picks from Around the Web (updated 28 JUL 2024 )
Old Science
Weird Science
Treating Disease with Fecal Transplantation
Fossils of miniature humans (hobbits) discovered in Indonesia
Paleontology
Dinosaur tail, complete with feathers, found preserved in amber.
Astronomy
Mysterious fast radio burst (FRB) detected in the distant universe.
Big Data & Informatics
Big Data: Buzzword or Big Deal?
Hacking the genome: Identifying anonymized human subjects using publicly available data.