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Bibliography on: CRISPR-Cas

The Electronic Scholarly Publishing Project: Providing world-wide, free access to classic scientific papers and other scholarly materials, since 1993.


ESP: PubMed Auto Bibliography 10 Dec 2023 at 01:43 Created: 


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: 2023-12-08
CmpDate: 2023-12-07

Kulhankova K, Traore S, Cheng X, et al (2023)

Shuttle peptide delivers base editor RNPs to rhesus monkey airway epithelial cells in vivo.

Nature communications, 14(1):8051.

Gene editing strategies for cystic fibrosis are challenged by the complex barrier properties of airway epithelia. We previously reported that the amphiphilic S10 shuttle peptide non-covalently combined with CRISPR-associated (Cas) ribonucleoprotein (RNP) enabled editing of human and mouse airway epithelial cells. Here, we derive the S315 peptide as an improvement over S10 in delivering base editor RNP. Following intratracheal aerosol delivery of Cy5-labeled peptide in rhesus macaques, we confirm delivery throughout the respiratory tract. Subsequently, we target CCR5 with co-administration of ABE8e-Cas9 RNP and S315. We achieve editing efficiencies of up-to 5.3% in rhesus airway epithelia. Moreover, we document persistence of edited epithelia for up to 12 months in mice. Finally, delivery of ABE8e-Cas9 targeting the CFTR R553X mutation restores anion channel function in cultured human airway epithelia. These results demonstrate the therapeutic potential of base editor delivery with S315 to functionally correct the CFTR R553X mutation in respiratory epithelia.

RevDate: 2023-12-08
CmpDate: 2023-12-07

Ramos A, Koch CE, Liu-Lupo Y, et al (2023)

Leukemia-intrinsic determinants of CAR-T response revealed by iterative in vivo genome-wide CRISPR screening.

Nature communications, 14(1):8048.

CAR-T therapy is a promising, novel treatment modality for B-cell malignancies and yet many patients relapse through a variety of means, including loss of CAR-T cells and antigen escape. To investigate leukemia-intrinsic CAR-T resistance mechanisms, we performed genome-wide CRISPR-Cas9 loss-of-function screens in an immunocompetent murine model of B-cell acute lymphoblastic leukemia (B-ALL) utilizing a modular guide RNA library. We identified IFNγR/JAK/STAT signaling and components of antigen processing and presentation pathway as key mediators of resistance to CAR-T therapy in vivo; intriguingly, loss of this pathway yielded the opposite effect in vitro (sensitized leukemia to CAR-T cells). Transcriptional characterization of this model demonstrated upregulation of these pathways in tumors relapsed after CAR-T treatment, and functional studies showed a surprising role for natural killer (NK) cells in engaging this resistance program. Finally, examination of data from B-ALL patients treated with CAR-T revealed an association between poor outcomes and increased expression of JAK/STAT and MHC-I in leukemia cells. Overall, our data identify an unexpected mechanism of resistance to CAR-T therapy in which tumor cell interaction with the in vivo tumor microenvironment, including NK cells, induces expression of an adaptive, therapy-induced, T-cell resistance program in tumor cells.

RevDate: 2023-12-08
CmpDate: 2023-12-07

Balke I, Silamikelis I, Radovica-Spalvina I, et al (2023)

Ryegrass mottle virus complete genome determination and development of infectious cDNA by combining two methods- 3' RACE and RNA-Seq.

PloS one, 18(12):e0287278.

Ryegrass mottle virus (RGMoV; genus: Sobemovirus) is a single-stranded positive RNA virus with a 30 nm viral particle size. It exhibits T = 3 symmetry with 180 coat protein (CP) subunits forming a viral structure. The RGMoV genome comprises five open reading frames that encode P1, Px, a membrane-anchored 3C-like serine protease, a viral genome-linked protein, P16, an RNA-dependent RNA polymerase, and CP. The RGMoV genome size varies, ranging from 4175 nt (MW411579.1) to 4253 nt (MW411579.1) in the deposited sequences. An earlier deposited RGMoV complete genome sequence of 4212 nt length (EF091714.1) was used to develop an infectious complementary DNA (icDNA) construct for in vitro gRNA transcription from the T7 promoter. However, viral infection was not induced when the transcribed gRNA was introduced into oat plants, indicating the potential absence of certain sequences in either the 5' or 3' untranslated regions (UTR) or both. The complete sequence of the 3' UTR was determined through 3' end RACE, while the 5' UTR was identified using high-throughput sequencing (HTS)-RNA-Seq to resolve the potential absences. Only the icDNA vector containing the newly identified UTR sequences proved infectious, resulting in typical viral infection symptoms and subsequent propagation of progeny viruses, exhibiting the ability to cause repeated infections in oat plants after at least one passage. The successful generation of icDNA highlighted the synergistic potential of utilizing both methods when a single approach failed. Furthermore, this study demonstrated the reliability of HTS as a method for determining the complete genome sequence of viral genomes.

RevDate: 2023-12-07
CmpDate: 2023-12-07

Malla RR, K Middela (2023)

CRISPR-Based Approaches for Cancer Immunotherapy.

Critical reviews in oncogenesis, 28(4):1-14.

Clustered regularly interspaced short palindromic repeats (CRISPR) technology is a powerful gene editing tool that has the potential to revolutionize cancer treatment. It allows for precise and efficient editing of specific genes that drive cancer growth and progression. CRISPR-based approaches gene knock-out, which deletes specific genes or sequences of DNA within a cancer cell, and gene knock-in, which inserts new sequences of DNA into a cancer cell to identify potential targets for cancer therapy. Further, genome-wide CRISPR-Cas9-based screens identify specific markers for diagnosis of cancers. Recently, immunotherapy has become a highly efficient strategy for the treatment of cancer. The use of CRISPR in cancer immunotherapy is focused on enhancing the function of T cells, making them more effective at attacking cancer cells and inactivating the immune evasion mechanisms of cancer cells. It has the potential to generate CAR-T cells, which are T cells that have been genetically engineered to target and attack cancer cells specifically. This review uncovers the latest developments in CRISPR-based gene editing strategies and delivery of their components in cancer cells. In addition, the applications of CRISPR in cancer immune therapy are discussed. Overall, this review helps to explore the potential of CRISPR-based strategies in cancer immune therapy in clinical settings.

RevDate: 2023-12-07
CmpDate: 2023-12-06

Tian X, Teo WFA, Wee WY, et al (2023)

Genome characterization and taxonomy of Actinomyces acetigenes sp. nov., and Actinomyces stomatis sp. nov., previously isolated from the human oral cavity.

BMC genomics, 24(1):734.

BACKGROUND: Actinomyces strains are commonly found as part of the normal microflora on human tissue surfaces, including the oropharynx, gastrointestinal tract, and female genital tract. Understanding the diversity and characterization of Actinomyces species is crucial for human health, as they play an important role in dental plaque formation and biofilm-related infections. Two Actinomyces strains ATCC 49340[ T] and ATCC 51655[ T] have been utilized in various studies, but their accurate species classification and description remain unresolved.

RESULTS: To investigate the genomic properties and taxonomic status of these strains, we employed both 16S rRNA Sanger sequencing and whole-genome sequencing using the Illumina HiSeq X Ten platform with PE151 (paired-end) sequencing. Our analyses revealed that the draft genome of Actinomyces acetigenes ATCC 49340[ T] was 3.27 Mbp with a 68.0% GC content, and Actinomyces stomatis ATCC 51655[ T] has a genome size of 3.08 Mbp with a 68.1% GC content. Multi-locus (atpA, rpoB, pgi, metG, gltA, gyrA, and core genome SNPs) sequence analysis supported the phylogenetic placement of strains ATCC 51655[ T] and ATCC 49340[ T] as independent lineages. Digital DNA-DNA hybridization (dDDH), average nucleotide identity (ANI), and average amino acid identity (AAI) analyses indicated that both strains represented novel Actinomyces species, with values below the threshold for species demarcation (70% dDDH, 95% ANI and AAI). Pangenome analysis identified 5,731 gene clusters with strains ATCC 49340[ T] and ATCC 51655[ T] possessing 1,515 and 1,518 unique gene clusters, respectively. Additionally, genomic islands (GIs) prediction uncovered 24 putative GIs in strain ATCC 49340[ T] and 16 in strain ATCC 51655[ T], contributing to their genetic diversity and potential adaptive capabilities. Pathogenicity analysis highlighted the potential human pathogenicity risk associated with both strains, with several virulence-associated factors identified. CRISPR-Cas analysis exposed the presence of CRISPR and Cas genes in both strains, indicating these strains might evolve a robust defense mechanism against them.

CONCLUSION: This study supports the classification of strains ATCC 49340[ T] and ATCC 51655[ T] as novel species within the Actinomyces, in which the name Actinomyces acetigenes sp. nov. (type strain ATCC 49340[ T] = VPI D163E-3[ T] = CCUG 34286[ T] = CCUG 35339 [T]) and Actinomyces stomatis sp. nov. (type strain ATCC 51655[ T] = PK606[T] = CCUG 33930[ T]) are proposed.

RevDate: 2023-12-07
CmpDate: 2023-12-06

Tao S, Hu C, Fang Y, et al (2023)

Targeted elimination of Vancomycin resistance gene vanA by CRISPR-Cas9 system.

BMC microbiology, 23(1):380.

OBJECTIVE: The purpose of this study is to reduce the spread of the vanA gene by curing the vanA-harboring plasmid of vancomycin-resistant using the CRISPR-Cas9 system.

METHODS: Two specific spacer sequence (sgRNAs) specific was designed to target the vanA gene and cloned into plasmid CRISPR-Cas9. The role of the CRISPR-Cas system in the plasmid elimination of drug-resistance genes was verified by chemically transformation and conjugation delivery methods. Moreover, the elimination efficiency in strains was evaluated by plate counting, PCR, and quantitative real-time PCR (qPCR). Susceptibility testing was performed by broth microdilution assay and by Etest strips (bioMérieux, France) to detect changes in bacterial drug resistance phenotype after drug resistance plasmid clearance.

RESULTS: In the study, we constructed a specific prokaryotic CRISPR-Cas9 system plasmid targeting cleavage of the vanA gene. PCR and qPCR results indicated that recombinant pCas9-sgRNA plasmid can efficiently clear vanA-harboring plasmids. There was no significant correlation between sgRNA lengths and curing efficiency. In addition, the drug susceptibility test results showed that the bacterial resistance to vancomycin was significantly reduced after the vanA-containing drug-resistant plasmid was specifically cleaved by the CRISPR-Cas system. The CRISPR-Cas9 system can block the horizontal transfer of the conjugated plasmid pUC19-vanA.

CONCLUSION: In conclusion, our study demonstrated that CRISPR-Cas9 achieved plasmid clearance and reduced antimicrobial resistance. The CRISPR-Cas9 system could block the horizontal transfer of plasmid carrying vanA. This strategy provided a great potential to counteract the ever-worsening spread of the vanA gene among bacterial pathogens and laid the foundation for subsequent research using the CRISPR-Cas9 system as adjuvant antibiotic therapy.

RevDate: 2023-12-07
CmpDate: 2023-12-06

Yin DP, Zhang H, Teng H, et al (2023)

Overexpressed Gαi1 exerts pro-tumorigenic activity in nasopharyngeal carcinoma.

Cell death & disease, 14(12):792.

The current study tested the expression and potential functions of Gαi1 in nasopharyngeal carcinoma (NPC). The Cancer Genome Atlas (TCGA) database results demonstrate that Gαi1 transcripts' number in NPC tissues is significantly higher than that in the normal nasal epithelial tissues. Its overexpression correlates with poor survival in certain NPC patients. Moreover, Gαi1 is significantly upregulated in NPC tissues of local primary patients and in different primary human NPC cells. Whereas its expression is relatively low in cancer-surrounding normal tissues and in primary nasal epithelial cells. Genetic silencing (via shRNA strategy) or knockout (via CRISPR-sgRNA method) of Gαi1 substantially suppressed viability, proliferation, cell cycle progression, and migration in primary NPC cells, causing significant caspase-apoptosis activation. Contrarily, ectopic Gαi1 expression exerted pro-tumorigenic activity and strengthened cell proliferation and migration in primary NPC cells. Gαi1 is important for Akt-mTOR activation in NPC cells. Akt-S6K phosphorylation was downregulated after Gαi1 shRNA or KO in primary NPC cells, but strengthened following Gαi1 overexpression. In Gαi1-silenced primary NPC cells, a S473D constitutively-active mutant Akt1 (caAkt1) restored Akt-S6K phosphorylation and ameliorated Gαi1 shRNA-induced proliferation inhibition, migration reduction and apoptosis. Bioinformatics analyses proposed zinc finger protein 384 (ZNF384) as a potential transcription factor of Gαi1. In primary NPC cells, ZNF384 shRNA or knockout (via CRISPR-sgRNA method) decreased Gαi1 mRNA and protein expression, whereas ZNF384 overexpression upregulated it. Importantly, there was an increased binding between ZNF384 protein and the Gαi1 promoter in human NPC tissues and different NPC cells. In vivo studies showed that intratumoral injection of Gαi1-shRNA-expressing adeno-associated virus (AAV) impeded subcutaneous NPC xenograft growth in nude mice. Gαi1 downregulation, Akt-mTOR inactivation, and apoptosis induction were detected in Gαi1-silenced NPC xenograft tissues. Gαi1 KO also effectively inhibited the growth of NPC xenografts in nude mice. Together, overexpressed Gαi1 exerts pro-tumorigenic activity in NPC possibly by promoting Akt-mTOR activation.

RevDate: 2023-12-07
CmpDate: 2023-12-06

Feng W, Bogomolovas J, Wang L, et al (2023)

ALPK3 Functions as a Pseudokinase.

Circulation, 148(23):1911-1913.

RevDate: 2023-12-05

Störtz F, Mak JK, P Minary (2023)

piCRISPR: Physically informed deep learning models for CRISPR/Cas9 off-target cleavage prediction.

Artificial intelligence in the life sciences, 3:None.

CRISPR/Cas programmable nuclease systems have become ubiquitous in the field of gene editing. With progressing development, applications in in vivo therapeutic gene editing are increasingly within reach, yet limited by possible adverse side effects from unwanted edits. Recent years have thus seen continuous development of off-target prediction algorithms trained on in vitro cleavage assay data gained from immortalised cell lines. It has been shown that in contrast to experimental epigenetic features, computed physically informed features are so far underutilised despite bearing considerably larger correlation with cleavage activity. Here, we implement state-of-the-art deep learning algorithms and feature encodings for off-target prediction with emphasis on physically informed features that capture the biological environment of the cleavage site, hence terming our approach piCRISPR. Features were gained from the large, diverse crisprSQL off-target cleavage dataset. We find that our best-performing models highlight the importance of sequence context and chromatin accessibility for cleavage prediction and compare favourably with literature standard prediction performance. We further show that our novel, environmentally sensitive features are crucial to accurate prediction on sequence-identical locus pairs, making them highly relevant for clinical guide design. The source code and trained models can be found ready to use at

RevDate: 2023-12-02

Wang L, Cao S, Li L, et al (2023)

Establishment of an ultrasensitive and visual detection platform for Neospora caninum based-on the RPA-CRISPR/Cas12a system.

Talanta, 269:125413 pii:S0039-9140(23)01164-5 [Epub ahead of print].

Neospora caninum is a protozoan parasite that causes neosporosis in cattle, and leads to a high rate of abortion and severe financial losses. Rapid and accurate detection is particularly important for preventing and controlling neosporosis. In our research, a highly effective diagnostic technique based on the RPA-CRISPR/Cas system was created to successfully identify N. caninum against the Nc5 gene, fluorescent reporter system and the lateral flow strip (LFS) biosensor were exploited to display results. The specificity and sensitivity of the PRA-CRISPR/Cas12a assay were evaluated. We discovered that it was highly specific and did not react with any other pathogens. The limit of detection (LOD) for this technology was as low as one parasite per milliliter when employing the fluorescent reporter system, and was approximately ten parasites per milliliter based on the LFS biosensor and under blue or UV light. Meanwhile, the placental tissue samples were detected by our RPA-CRISPR/Cas12a detection platform were completely consistent with that of the nested PCR assay (59.4 %, 19/32). The canine feces were detected by our RPA-CRISPR/Cas12a detection platform were completely consistent with that of the nested PCR assay (8.6 %, 6/70). The RPA-CRISPR/Cas12a detection procedure was successfully finished in within 90 min and offers advantages of high sensitivity and specificity, speed and low cost. The technique was better suitable for extensive neosporosis screening in non-laboratory and resource-constrained locations. This study provided a new strategy for more rapid and portable identification of N. caninum.

RevDate: 2023-12-02

Schmitz M, I Querques (2023)

DNA on the move: mechanisms, functions and applications of transposable elements.

FEBS open bio [Epub ahead of print].

Transposons are mobile genetic elements that have invaded all domains of life by moving between and within their host genomes. Due to their mobility (or transposition), transposons facilitate horizontal gene transfer in bacteria and foster the evolution of new molecular functions in prokaryotes and eukaryotes. As transposition can lead to detrimental genomic rearrangements, organisms have evolved a multitude of molecular strategies to control transposons, including genome defence mechanisms provided by CRISPR-Cas systems. Apart from their biological impacts on genomes, DNA transposons have been leveraged as efficient gene insertion vectors in basic research, transgenesis and gene therapy. However, the close to random insertion profile of transposon-based tools limits their programmability and safety. Despite recent advances brought by the development of CRISPR-associated genome editing nucleases, a strategy for efficient insertion of large, multi-kilobase transgenes at user-defined genomic sites is currently challenging. The discovery and experimental characterization of bacterial CRISPR-associated transposons (CASTs) led to the attractive hypothesis that these systems could be repurposed as programmable, site-specific gene integration technologies. Here, we provide a broad overview of the molecular mechanisms underpinning DNA transposition and of its biological and technological impact. The second focus of the article is to describe recent mechanistic and functional analyses of CAST transposition. Finally, current challenges and desired future advances of CAST-based genome engineering applications are briefly discussed.

RevDate: 2023-12-05
CmpDate: 2023-12-04

Zhang J, Xu Y, Wang M, et al (2023)

Mobilizable plasmids drive the spread of antimicrobial resistance genes and virulence genes in Klebsiella pneumoniae.

Genome medicine, 15(1):106.

BACKGROUND: Klebsiella pneumoniae is a notorious clinical pathogen and frequently carries various plasmids, which are the main carriers of antimicrobial resistance and virulence genes. In comparison to self-transmissible conjugative plasmids, mobilizable plasmids have received much less attention due to their defects in conjugative elements. However, the contribution of mobilizable plasmids to the horizontal transfer of antimicrobial resistance genes and virulence genes of K. pneumoniae remains unclear. In this study, the transfer, stability, and cargo genes of the mobilizable plasmids of K. pneumoniae were examined via genetic experiments and genomic analysis.

METHODS: Carbapenem-resistant (CR) plasmid pHSKP2 and multidrug-resistant (MDR) plasmid pHSKP3 of K. pneumoniae HS11286, virulence plasmid pRJF293 of K. pneumoniae RJF293 were employed in conjugation assays to assess the transfer ability of mobilizable plasmids. Mimic mobilizable plasmids and genetically modified plasmids were constructed to confirm the cotransfer models. The plasmid morphology was evaluated through XbaI and S1 nuclease pulsed-field gel electrophoresis and/or complete genome sequencing. Mobilizable plasmid stability in transconjugants was analyzed via serial passage culture. In addition, in silico genome analysis of 3923 plasmids of 1194 completely sequenced K. pneumoniae was performed to investigate the distribution of the conjugative elements, the cargo genes, and the targets of the CRISPR-Cas system. The mobilizable MDR plasmid and virulence plasmid of K. pneumoniae were investigated, which carry oriT but lack other conjugative elements.

RESULTS: Our results showed that mobilizable MDR and virulence plasmids carrying oriT but lacking the relaxase gene were able to cotransfer with a helper conjugative CR plasmid across various Klebsiella and Escherichia coli strains. The transfer and stability of mobilizable plasmids rather than conjugative plasmids were not interfered with by the CRISPR-Cas system of recipient strains. According to the in silico analysis, the mobilizable plasmids carry about twenty percent of acquired antimicrobial resistance genes and more than seventy-five percent of virulence genes in K. pneumoniae.

CONCLUSIONS: Our work observed that a mobilizable MDR or virulence plasmid that carries oriT but lacks the relaxase genes transferred with the helper CR conjugative plasmid and mobilizable plasmids escaped from CRISPR-Cas defence and remained stable in recipients. These results highlight the threats of mobilizable plasmids as vital vehicles in the dissemination of antibiotic resistance and virulence genes in K. pneumoniae.

RevDate: 2023-12-04
CmpDate: 2023-12-04

Cavanaugh C, Hesson J, J Mathieu (2024)

Genomic Engineering of Induced Pluripotent Stem Cell-Derived Cardiomyocytes.

Methods in molecular biology (Clifton, N.J.), 2735:129-143.

Recent advances in patient-derived induced Pluripotent Stem Cell (iPSC) generation, improvement of cardiomyocyte-directed differentiation protocols, and the availability of new genome editing techniques have opened up new avenues for disease modeling of cardiomyopathies. Patients with cardiomyopathies often harbor a single-base substitution believed to be linked to the disease phenotype. Somatic cells derived from patients can be efficiently reprogrammed into iPSCs and subsequently engineered. The targeting of a precise mutation can be achieved by the introduction of double stranded breaks with CRISPR-Cas9 and by homology-directed repair when using a DNA donor template. This allows for the correction of a mutation in a patient iPSC line to generate an isogenic control. In addition, key mutations associated with cardiomyopathies can be introduced in an iPSC line derived from a healthy individual using the same techniques. In this chapter, we describe in detail how to engineer pluripotent stem cells to model cardiomyopathy in a dish using CRISPR-Cas9 technology.

RevDate: 2023-11-30

Liu R, Yao J, Zhou S, et al (2023)

Spatiotemporal control of RNA metabolism and CRISPR-Cas functions using engineered photoswitchable RNA-binding proteins.

Nature protocols [Epub ahead of print].

RNA molecules perform various crucial roles in diverse cellular processes, from translating genetic information to decoding the genome, regulating gene expression and catalyzing chemical reactions. RNA-binding proteins (RBPs) play an essential role in regulating the diverse behaviors and functions of RNA in live cells, but techniques for the spatiotemporal control of RBP activities and RNA functions are rarely reported yet highly desirable. We recently reported the development of LicV, a synthetic photoswitchable RBP that can bind to a specific RNA sequence in response to blue light irradiation. LicV has been used successfully for the optogenetic control of RNA localization, splicing, translation and stability, as well as for the photoswitchable regulation of transcription and genomic locus labeling. Compared to classical genetic or pharmacologic perturbations, LicV-based light-switchable effectors have the advantages of large dynamic range between dark and light conditions and submicron and millisecond spatiotemporal resolutions. In this protocol, we provide an easy, efficient and generalizable strategy for engineering photoswitchable RBPs for the spatiotemporal control of RNA metabolism. We also provide a detailed protocol for the conversion of a CRISPR-Cas system to optogenetic control. The protocols typically take 2-3 d, including transfection and results analysis. Most of this protocol is applicable to the development of novel LicV-based photoswitchable effectors for the optogenetic control of other RNA metabolisms and CRISPR-Cas functions.

RevDate: 2023-11-30

Becker HJ, S Yamazaki (2023)

Understanding Genetic Heterogeneity in Gene-Edited HSC Products.

Experimental hematology pii:S0301-472X(23)01770-8 [Epub ahead of print].

CRISPR/Cas gene editing has transformed genetic research and is poised to drive the next generation of gene therapies targeting hematopoietic stem cells (HSCs). However, the installation of the 'desired' edit is most often only achieved in a minor subset of alleles. The array of cellular pathways triggered by gene editing tools produces a broad spectrum of 'undesired' editing outcomes, including short insertions and deletions (indels) and chromosome rearrangements, leading to considerable genetic heterogeneity in gene edited HSC populations. This heterogeneity may undermine the effect of the genetic intervention, since only a subset of cells will carry the intended modification. Also, undesired mutations represent a potential safety concern as gene editing advances toward broader clinical use. Here, we will review the different sources of 'undesired' edits and will discuss strategies for their mitigation and control.

RevDate: 2023-12-02

Nadi R, Juan-Vicente L, Mateo-Bonmatí E, et al (2023)

The unequal functional redundancy of Arabidopsis INCURVATA11 and CUPULIFORMIS2 is not dependent on genetic background.

Frontiers in plant science, 14:1239093.

The paralogous genes INCURVATA11 (ICU11) and CUPULIFORMIS2 (CP2) encode components of the epigenetic machinery in Arabidopsis and belong to the 2-oxoglutarate and Fe (II)-dependent dioxygenase superfamily. We previously inferred unequal functional redundancy between ICU11 and CP2 from a study of the synergistic phenotypes of the double mutant and sesquimutant combinations of icu11 and cp2 mutations, although they represented mixed genetic backgrounds. To avoid potential confounding effects arising from different genetic backgrounds, we generated the icu11-5 and icu11-6 mutants via CRISPR/Cas genome editing in the Col-0 background and crossed them to cp2 mutants in Col-0. The resulting mutants exhibited a postembryonic-lethal phenotype reminiscent of strong embryonic flower (emf) mutants. Double mutants involving icu11-5 and mutations affecting epigenetic machinery components displayed synergistic phenotypes, whereas cp2-3 did not besides icu11-5. Our results confirmed the unequal functional redundancy between ICU11 and CP2 and demonstrated that it is not allele or genetic background specific. An increase in sucrose content in the culture medium partially rescued the post-germinative lethality of icu11 cp2 double mutants and sesquimutants, facilitating the study of their morphological phenotypes throughout their life cycle, which include floral organ homeotic transformations. We thus established that the ICU11-CP2 module is required for proper flower organ identity.

RevDate: 2023-12-02

Cavazza A, Hendel A, Bak RO, et al (2023)

Progress and harmonization of gene editing to treat human diseases: Proceeding of COST Action CA21113 GenE-HumDi.

Molecular therapy. Nucleic acids, 34:102066.

The European Cooperation in Science and Technology (COST) is an intergovernmental organization dedicated to funding and coordinating scientific and technological research in Europe, fostering collaboration among researchers and institutions across countries. Recently, COST Action funded the "Genome Editing to treat Human Diseases" (GenE-HumDi) network, uniting various stakeholders such as pharmaceutical companies, academic institutions, regulatory agencies, biotech firms, and patient advocacy groups. GenE-HumDi's primary objective is to expedite the application of genome editing for therapeutic purposes in treating human diseases. To achieve this goal, GenE-HumDi is organized in several working groups, each focusing on specific aspects. These groups aim to enhance genome editing technologies, assess delivery systems, address safety concerns, promote clinical translation, and develop regulatory guidelines. The network seeks to establish standard procedures and guidelines for these areas to standardize scientific practices and facilitate knowledge sharing. Furthermore, GenE-HumDi aims to communicate its findings to the public in accessible yet rigorous language, emphasizing genome editing's potential to revolutionize the treatment of many human diseases. The inaugural GenE-HumDi meeting, held in Granada, Spain, in March 2023, featured presentations from experts in the field, discussing recent breakthroughs in delivery methods, safety measures, clinical translation, and regulatory aspects related to gene editing.

RevDate: 2023-11-30

Jungfer K, Sigg A, M Jinek (2023)

Substrate selectivity and catalytic activation of the type III CRISPR ancillary nuclease Can2.

Nucleic acids research pii:7456044 [Epub ahead of print].

Type III CRISPR-Cas systems provide adaptive immunity against foreign mobile genetic elements through RNA-guided interference. Sequence-specific recognition of RNA targets by the type III effector complex triggers the generation of cyclic oligoadenylate (cOA) second messengers that activate ancillary effector proteins, thus reinforcing the host immune response. The ancillary nuclease Can2 is activated by cyclic tetra-AMP (cA4); however, the mechanisms underlying cA4-mediated activation and substrate selectivity remain elusive. Here we report crystal structures of Thermoanaerobacter brockii Can2 (TbrCan2) in substrate- and product-bound complexes. We show that TbrCan2 is a single strand-selective DNase and RNase that binds substrates via a conserved SxTTS active site motif, and reveal molecular interactions underpinning its sequence preference for CA dinucleotides. Furthermore, we identify a molecular interaction relay linking the cA4 binding site and the nuclease catalytic site to enable divalent metal cation coordination and catalytic activation. These findings provide key insights into the molecular mechanisms of Can2 nucleases in type III CRISPR-Cas immunity and may guide their technological development for nucleic acid detection applications.

RevDate: 2023-12-07
CmpDate: 2023-12-07

Reardon S (2023)

'Treasure trove' of new CRISPR systems holds promise for genome editing.

Nature, 624(7990):17-18.

RevDate: 2023-12-07
CmpDate: 2023-12-07

Kim EP, Kim DY, Park C, et al (2023)

Effects of klotho protein or klotho knockdown in porcine oocytes at different stages.

Zygote (Cambridge, England), 31(6):577-581.

Klotho is a protein that plays different functions in female fertility. We have previously reported that klotho protein supplementation during in vitro maturation improves porcine embryo development, while klotho knockout for somatic cell cloning completely blocks full-term pregnancy in vivo. However, the effects of the microinjection of klotho protein or klotho knockdown dual vector in porcine embryos at different time points and the specific molecular mechanisms remain largely unknown. In this study, we injected the preassembled cas9 + sgRNA dual vector, for klotho knockdown, into the cytoplasm of the germinal vesicle stage of oocytes and into porcine embryos after 6-h parthenogenetic activation. Similarly, the klotho protein was inserted into the cytoplasm of germinal vesicle stage oocytes and porcine embryos after 6-h parthenogenetic activation. Compared with the controls, the microinjection of klotho dual vector markedly decreased the blastocyst formation rates in germinal vesicle stage oocytes and activated embryos. However, the efficiency of blastocyst formation when klotho protein was inserted before in vitro maturation was significantly higher than that after klotho protein insertion into parthenogenetically activated embryos. These results indicated that klotho knockdown may impair embryo development into blastocyst irrespective of injection timing. In addition, klotho protein injection timing in pig embryos may be an important factor for regulating embryo development.

RevDate: 2023-12-07
CmpDate: 2023-12-07

Khanna K, Ohri P, R Bhardwaj (2023)

Nanotechnology and CRISPR/Cas9 system for sustainable agriculture.

Environmental science and pollution research international, 30(56):118049-118064.

The clustered regularly interspaced short palindromic repeats (CRISPR-Cas9), a genome editing tool, has gained a tremendous position due to its therapeutic efficacy, ability to counteract abiotic/biotic stresses in plants, environmental remediation and sustainable agriculture with the aim of food security. This is mainly due to their potential of precised genome modification and numerous genetic engineering protocols with versatility as well as simplicity. This technique is quite useful for crop refinement and overcoming the agricultural losses and regaining the soil fertility hampered by hazardous chemicals. Since CRISPR/Cas9 has been widely accepted in genome editing in plants, however, their revolutionised nature and progress enable genetic engineers to face numerous challenges in plant biotechnology. Therefore, nanoparticles have addressed these challenges and improved cargo delivery and genomic editing processes. Henceforth, this barrier prevents CRISPR-based genetic engineering in plants in order to show efficacy in full potential and eliminate all the barriers. This advancement accelerates the genome editing process and its applications in plant biotechnology enable us to sustain and feed the massive population under varying environments. Genome editing tools using CRISPR/Cas9 and nanotechnology are advantageous that produce transgenic-free plants that overcome global food demands. Here, in this review, we have aimed towards the mechanisms/delivery systems linked with CRISPR/Cas9 system. We have elaborated on the applications of CRISPR/Cas9 and nanotechnology-based systems for sustainable agriculture. Moreover, the challenges and limitations associated with genome editing and delivery systems have also been discussed with a special emphasis on crop improvement.

RevDate: 2023-12-02

Sinha S, Molina Vargas AM, Arantes PR, et al (2023)

Unveiling the RNA-mediated allosteric activation discloses functional hotspots in CRISPR-Cas13a.

Nucleic acids research pii:7456045 [Epub ahead of print].

Cas13a is a recent addition to the CRISPR-Cas toolkit that exclusively targets RNA, which makes it a promising tool for RNA detection. It utilizes a CRISPR RNA (crRNA) to target RNA sequences and trigger a composite active site formed by two 'Higher Eukaryotes and Prokaryotes Nucleotide' (HEPN) domains, cleaving any solvent-exposed RNA. In this system, an intriguing form of allosteric communication controls the RNA cleavage activity, yet its molecular details are unknown. Here, multiple-microsecond molecular dynamics simulations are combined with graph theory to decipher this intricate activation mechanism. We show that the binding of a target RNA acts as an allosteric effector, by amplifying the communication signals over the dynamical noise through interactions of the crRNA at the buried HEPN1-2 interface. By introducing a novel Signal-to-Noise Ratio (SNR) of communication efficiency, we reveal critical allosteric residues-R377, N378, and R973-that rearrange their interactions upon target RNA binding. Alanine mutation of these residues is shown to select target RNA over an extended complementary sequence beyond guide-target duplex for RNA cleavage, establishing the functional significance of these hotspots. Collectively our findings offer a fundamental understanding of the Cas13a mechanism of action and pave new avenues for the development of highly selective RNA-based cleavage and detection tools.

RevDate: 2023-12-04
CmpDate: 2023-12-04

Mogila I, Tamulaitiene G, Keda K, et al (2023)

Ribosomal stalk-captured CARF-RelE ribonuclease inhibits translation following CRISPR signaling.

Science (New York, N.Y.), 382(6674):1036-1041.

Prokaryotic type III CRISPR-Cas antiviral systems employ cyclic oligoadenylate (cAn) signaling to activate a diverse range of auxiliary proteins that reinforce the CRISPR-Cas defense. Here we characterize a class of cAn-dependent effector proteins named CRISPR-Cas-associated messenger RNA (mRNA) interferase 1 (Cami1) consisting of a CRISPR-associated Rossmann fold sensor domain fused to winged helix-turn-helix and a RelE-family mRNA interferase domain. Upon activation by cyclic tetra-adenylate (cA4), Cami1 cleaves mRNA exposed at the ribosomal A-site thereby depleting mRNA and leading to cell growth arrest. The structures of apo-Cami1 and the ribosome-bound Cami1-cA4 complex delineate the conformational changes that lead to Cami1 activation and the mechanism of Cami1 binding to a bacterial ribosome, revealing unexpected parallels with eukaryotic ribosome-inactivating proteins.

RevDate: 2023-12-01
CmpDate: 2023-12-01

Nagorska A, Zaucker A, Lambert F, et al (2023)

Translational control of furina by an RNA regulon is important for left-right patterning, heart morphogenesis and cardiac valve function.

Development (Cambridge, England), 150(23):.

Heart development is a complex process that requires asymmetric positioning of the heart, cardiac growth and valve morphogenesis. The mechanisms controlling heart morphogenesis and valve formation are not fully understood. The pro-convertase FurinA functions in heart development across vertebrates. How FurinA activity is regulated during heart development is unknown. Through computational analysis of the zebrafish transcriptome, we identified an RNA motif in a variant FurinA transcript harbouring a long 3' untranslated region (3'UTR). The alternative 3'UTR furina isoform is expressed prior to organ positioning. Somatic deletions in the furina 3'UTR lead to embryonic left-right patterning defects. Reporter localisation and RNA-binding assays show that the furina 3'UTR forms complexes with the conserved RNA-binding translational repressor, Ybx1. Conditional ybx1 mutant embryos show premature and increased Furin reporter expression, abnormal cardiac morphogenesis and looping defects. Mutant ybx1 hearts have an expanded atrioventricular canal, abnormal sino-atrial valves and retrograde blood flow from the ventricle to the atrium. This is similar to observations in humans with heart valve regurgitation. Thus, the furina 3'UTR element/Ybx1 regulon is important for translational repression of FurinA and regulation of heart development.

RevDate: 2023-12-01

Misra V, Mall AK, Pandey H, et al (2023)

Advancements and prospects of CRISPR/Cas9 technologies for abiotic and biotic stresses in sugar beet.

Frontiers in genetics, 14:1235855.

Sugar beet is a crop with high sucrose content, known for sugar production and recently being considered as an emerging raw material for bioethanol production. This crop is also utilized as cattle feed, mainly when animal green fodder is scarce. Bioethanol and hydrogen gas production from this crop is an essential source of clean energy. Environmental stresses (abiotic/biotic) severely affect the productivity of this crop. Over the past few decades, the molecular mechanisms of biotic and abiotic stress responses in sugar beet have been investigated using next-generation sequencing, gene editing/silencing, and over-expression approaches. This information can be efficiently utilized through CRISPR/Cas 9 technology to mitigate the effects of abiotic and biotic stresses in sugar beet cultivation. This review highlights the potential use of CRISPR/Cas 9 technology for abiotic and biotic stress management in sugar beet. Beet genes known to be involved in response to alkaline, cold, and heavy metal stresses can be precisely modified via CRISPR/Cas 9 technology for enhancing sugar beet's resilience to abiotic stresses with minimal off-target effects. Similarly, CRISPR/Cas 9 technology can help generate insect-resistant sugar beet varieties by targeting susceptibility-related genes, whereas incorporating Cry1Ab and Cry1C genes may provide defense against lepidopteron insects. Overall, CRISPR/Cas 9 technology may help enhance sugar beet's adaptability to challenging environments, ensuring sustainable, high-yield production.

RevDate: 2023-12-01

Budzko L, Hoffa-Sobiech K, Jackowiak P, et al (2023)

Engineered deaminases as a key component of DNA and RNA editing tools.

Molecular therapy. Nucleic acids, 34:102062.

Over recent years, zinc-dependent deaminases have attracted increasing interest as key components of nucleic acid editing tools that can generate point mutations at specific sites in either DNA or RNA by combining a targeting module (such as a catalytically impaired CRISPR-Cas component) and an effector module (most often a deaminase). Deaminase-based molecular tools are already being utilized in a wide spectrum of therapeutic and research applications; however, their medical and biotechnological potential seems to be much greater. Recent reports indicate that the further development of nucleic acid editing systems depends largely on our ability to engineer the substrate specificity and catalytic activity of the editors themselves. In this review, we summarize the current trends and achievements in deaminase engineering. The presented data indicate that the potential of these enzymes has not yet been fully revealed or understood. Several examples show that even relatively minor changes in the structure of deaminases can give them completely new and unique properties.

RevDate: 2023-12-06
CmpDate: 2023-12-06

Ma YF, Zhang MQ, Gong LL, et al (2023)

Efficient nanoparticle-based CRISPR-Cas13d induced mRNA disruption of an eye pigmentation gene in the white-backed planthopper, Sogatella furcifera.

Insect science, 30(6):1552-1564.

The discovery of the clustered regularly interspaced short palindromic repeat (CRISPR) system has driven gene manipulation technology to a new era with applications reported in organisms that span the tree of life. The utility of CRISPR-mediated editing was further expanded to mRNA following identification of the RNA-targeting Cas13 family of smaller endonuclease proteins. Application of this family to insect research, however, has been more limited. In this study, the smallest Cas13 family member, Cas13d, and guide RNAs (gRNAs) were complexed with a versatile nanomaterial (star polycation, SPc) to generate a proof-of-concept RNA-editing platform capable of disrupting mRNA expression of the eye pigmentation gene tryptophan 2,3-dioxygenase (SfTO) in white-backed planthoppers (WBPHs). The resulting red-eye phenotype was present in 19.76% (with SPc) and 22.99% (without SPc) of the treatment groups and was comparable to the red-eye phenotype generated following conventional RNA interference knockdown (22.22%). Furthermore, the Cas13/gRNA phenotype manifested more quickly than RNA interference. Consistent with the expected Cas13d mechanism, SfTO transcript levels were significantly reduced. Taken together, the results indicate that the SPc-CRISPR-Cas13d/gRNA complex negatively impacted expression of the target gene. These findings confirm the utility of this novel mRNA disruption system in insects and lay the foundation for further development of these tools in the implementation of green agricultural pest management tactics.

RevDate: 2023-12-05
CmpDate: 2023-12-05

Bi M, Wang Z, Cheng K, et al (2023)

Construction of transcription factor mutagenesis population in tomato using a pooled CRISPR/Cas9 plasmid library.

Plant physiology and biochemistry : PPB, 205:108094.

Adequate mutant materials are the prerequisite for conducting gene function research or screening novel functional genes in plants. The strategy of constructing a large-scale mutant population using the pooled CRISPR/Cas9-sgRNA library has been implemented in several crops. However, the effective application of this CRISPR/Cas9 large-scale screening strategy to tomato remains to be attempted. Here, we identified 990 transcription factors in the tomato genome, designed and synthesized a CRISPR/Cas9 plasmid library containing 4379 sgRNAs. Using this pooled library, 487 T0 positive plants were obtained, among which 92 plants harbored a single sgRNA sequence, targeting 65 different transcription factors, with a mutation rate of 23%. In the T0 mutant population, the occurrence of homozygous and biallelic mutations was observed at higher frequencies. Additionally, the utilization of a small-scale CRISPR/Cas9 library targeting 30 transcription factors could enhance the efficacy of single sgRNA recognition in positive plants, increasing it from 19% to 42%. Phenotypic characterization of several mutants identified from the mutant population demonstrated the utility of our CRISPR/Cas9 mutant library. Taken together, our study offers insights into the implementation and optimization of CRISPR/Cas9-mediated large-scale knockout library in tomato.

RevDate: 2023-12-05
CmpDate: 2023-12-05

Papkou A, Garcia-Pastor L, Escudero JA, et al (2023)

A rugged yet easily navigable fitness landscape.

Science (New York, N.Y.), 382(6673):eadh3860.

Fitness landscape theory predicts that rugged landscapes with multiple peaks impair Darwinian evolution, but experimental evidence is limited. In this study, we used genome editing to map the fitness of >260,000 genotypes of the key metabolic enzyme dihydrofolate reductase in the presence of the antibiotic trimethoprim, which targets this enzyme. The resulting landscape is highly rugged and harbors 514 fitness peaks. However, its highest peaks are accessible to evolving populations via abundant fitness-increasing paths. Different peaks share large basins of attraction that render the outcome of adaptive evolution highly contingent on chance events. Our work shows that ruggedness need not be an obstacle to Darwinian evolution but can reduce its predictability. If true in general, the complexity of optimization problems on realistic landscapes may require reappraisal.

RevDate: 2023-12-05

Bell RT, Sahakyan H, Makarova KS, et al (2023)

CoCoNuTs: A diverse subclass of Type IV restriction systems predicted to target RNA.

bioRxiv : the preprint server for biology pii:2023.07.31.551357.

A comprehensive census of McrBC systems, among the most common forms of prokaryotic Type IV restriction systems, followed by phylogenetic analysis, reveals their enormous abundance in diverse prokaryotes and a plethora of genomic associations. We focus on a previously uncharacterized branch, which we denote CoCoNuTs (coiled-coil nuclease tandems) for their salient features: the presence of extensive coiled-coil structures and tandem nucleases. The CoCoNuTs alone show extraordinary variety, with 3 distinct types and multiple subtypes. All CoCoNuTs contain domains predicted to interact with translation system components, such as OB-folds resembling the SmpB protein that binds bacterial tmRNA, YTH-like domains that might recognize methylated tmRNA, tRNA, or rRNA, and RNA-binding Hsp70 chaperone homologs, along with RNases, such as HEPN domains, all suggesting that the CoCoNuTs target RNA. Many CoCoNuTs might additionally target DNA, via McrC nuclease homologs. Additional restriction systems, such as Type I RM, BREX, and Druantia Type III, are frequently encoded in the same predicted superoperons. In many of these superoperons, CoCoNuTs are likely regulated by cyclic nucleotides, possibly, RNA fragments with cyclic termini, that bind associated CARF (CRISPR-Associated Rossmann Fold) domains. The CoCoNuTs, together with the ancillary restriction factors, might employ an echeloned defense strategy analogous to that of Type III CRISPR-Cas systems, in which an immune response eliminating virus DNA and/or RNA is launched first, but then, if it fails, an abortive infection response leading to PCD/dormancy via host RNA cleavage takes over.

RevDate: 2023-12-05
CmpDate: 2023-12-05

Liao T, Xu X, Wu J, et al (2023)

Increased expression levels of DLX5 inhibit the development of the nervous system.

International journal of developmental neuroscience : the official journal of the International Society for Developmental Neuroscience, 83(8):728-739.

INTRODUCTION: Preeclampsia is a hypertensive disorder of pregnancy. DLX5 plays an important role in the migration and differentiation of subglobus pallidus precursor cells.

METHODS: We established a zebrafish line expressing high levels of DLX5 and investigated changes in behavior and development of the nervous system.

RESULTS: The ratios of brain volume area to whole body area at 96 hpf zebrafish in the experimental group (gRNA + CasRx) were significantly lower than the WT group and the negative control group (casRx) (P < 0.01). Behavioral trajectory distances and movement speeds exhibited by the 6th day of development in zebrafish in the experimental group (gRNA + CasRx) were significantly shorter (P < 0.01) and lower (P < 0.05) than the negative control group (gRNA + CasRx), respectively.

CONCLUSIONS: Data suggested that the increased expression levels of DLX5 can inhibit brain volume development and behavioral activities in zebrafish. Maybe the high expression levels of DLX5 in the pathological state of preeclampsia can inhibit the development of the nervous system in offspring.

RevDate: 2023-12-05
CmpDate: 2023-12-05

Martin PB, Holbrook SE, Hicks AN, et al (2023)

Clinically relevant mouse models of Charcot-Marie-Tooth type 2S.

Human molecular genetics, 32(8):1276-1288.

Charcot-Marie-Tooth disease is an inherited peripheral neuropathy that is clinically and genetically heterogenous. Mutations in IGHMBP2, a ubiquitously expressed DNA/RNA helicase, have been shown to cause the infantile motor neuron disease spinal muscular atrophy with respiratory distress type 1 (SMARD1), and, more recently, juvenile-onset Charcot-Marie-Tooth disease type 2S (CMT2S). Using CRISPR-cas9 mutagenesis, we developed the first mouse models of CMT2S [p.Glu365del (E365del) and p.Tyr918Cys (Y918C)]. E365del is the first CMT2S mouse model to be discovered and Y918C is the first human CMT2S allele knock-in model. Phenotypic characterization of the homozygous models found progressive peripheral motor and sensory axonal degeneration. Neuromuscular and locomotor assays indicate that both E365del and Y918C mice have motor deficits, while neurobehavioral characterization of sensory function found that E365del mutants have mechanical allodynia. Analysis of femoral motor and sensory nerves identified axonal degeneration, which does not impact nerve conduction velocities in E365del mice, but it does so in the Y918C model. Based on these results, the E365del mutant mouse, and the human allele knock-in, Y918C, represent mouse models with the hallmark phenotypes of CMT2S, which will be critical for understanding the pathogenic mechanisms of IGHMBP2. These mice will complement existing Ighmbp2 alleles modeling SMARD1 to help understand the complex phenotypic and genotypic heterogeneity that is observed in patients with IGHMBP2 variants.

RevDate: 2023-12-01

Suprasanna P, Klimek-Chodacka M, SM Jain (2023)

Editorial: CRISPR tools, technology development, and application.

Frontiers in plant science, 14:1329780.

RevDate: 2023-12-01

Zhang S, Wang Y, Mao D, et al (2023)

Current trends of clinical trials involving CRISPR/Cas systems.

Frontiers in medicine, 10:1292452.

The CRISPR/Cas9 system is a powerful genome editing tool that has made enormous impacts on next-generation molecular diagnostics and therapeutics, especially for genetic disorders that traditional therapies cannot cure. Currently, CRISPR-based gene editing is widely applied in basic, preclinical, and clinical studies. In this review, we attempt to identify trends in clinical studies involving CRISPR techniques to gain insights into the improvement and contribution of CRISPR/Cas technologies compared to traditional modified modalities. The review of clinical trials is focused on the applications of the CRISPR/Cas systems in the treatment of cancer, hematological, endocrine, and immune system diseases, as well as in diagnostics. The scientific basis underlined is analyzed. In addition, the challenges of CRISPR application in disease therapies and recent advances that expand and improve CRISPR applications in precision medicine are discussed.

RevDate: 2023-12-01
CmpDate: 2023-12-01

Maltseva EA, Vasil'eva IA, Moor NA, et al (2023)

Cas9 is mostly orthogonal to human systems of DNA break sensing and repair.

PloS one, 18(11):e0294683.

CRISPR/Cas9 system is а powerful gene editing tool based on the RNA-guided cleavage of target DNA. The Cas9 activity can be modulated by proteins involved in DNA damage signalling and repair due to their interaction with double- and single-strand breaks (DSB and SSB, respectively) generated by wild-type Cas9 or Cas9 nickases. Here we address the interplay between Streptococcus pyogenes Cas9 and key DNA repair factors, including poly(ADP-ribose) polymerase 1 (SSB/DSB sensor), its closest homolog poly(ADP-ribose) polymerase 2, Ku antigen (DSB sensor), DNA ligase I (SSB sensor), replication protein A (DNA duplex destabilizer), and Y-box binding protein 1 (RNA/DNA binding protein). None of those significantly affected Cas9 activity, while Cas9 efficiently shielded DSBs and SSBs from their sensors. Poly(ADP-ribosyl)ation of Cas9 detected for poly(ADP-ribose) polymerase 2 had no apparent effect on the activity. In cellulo, Cas9-dependent gene editing was independent of poly(ADP-ribose) polymerase 1. Thus, Cas9 can be regarded as an enzyme mostly orthogonal to the natural regulation of human systems of DNA break sensing and repair.

RevDate: 2023-12-01
CmpDate: 2023-12-01

Blicharska D, Szućko-Kociuba I, Filip E, et al (2023)

[PRIME EDITING - a new method of editing genes].

Postepy biochemii, 69(3):146-158.

The Prime Editing method introduces the expected manipulations within a given genome with a Cas9-nicase and pegRNA structure and a reverse transcriptase, which is responsible for the synthesis of the segment, which is then incorporated into the edited strand. This technique is based on the previously discovered CRISPR/Cas9 method. It differs from CRISPR/Cas9 in the absence of double cracks within the DNA helix, which is due to its complex structure, including the presence of additional elements, i. e. the reverse transcriptase and the matrix within the pegRNA. PE is used to modify the DNA double helix. The work deals mainly with the creation and improvement as well as testing of the modern Prime Editing method. Information on the structure and functioning of the system is provided, as well as the research carried out so far with the use of PE, carried out within the genomes of cells derived from plant, animal, and human organisms, is described. The paper also contains information on the potential benefits and hopes related to the use of this innovative method.

RevDate: 2023-12-04
CmpDate: 2023-12-04

Zhang T, Xie Z, Zheng X, et al (2024)

CRISPR-Cas12a powered hybrid nanoparticle for extracellular vesicle aggregation and in-situ microRNA detection.

Biosensors & bioelectronics, 245:115856.

Efficient extracellular vesicle (EV) enrichment and timely internal RNA detection for cancer diagnostics are highly desirable and remain a challenge. Here, we report a rapid EV aggregation induced in-situ microRNA detection technology based on cationic lipid-polymer hybrid nanoparticles encapsulating cascade system of catalytic hairpin assembly and CRISPR-Cas12a (CLHN-CCC), allowing for EV enrichment in three-dimensional space and in-situ detection of internal microRNAs in one step within 30 min. The enrichment efficiency (>90%) of CLHN-CCC is demonstrated in artificial EVs, cell-secreted EVs and serum EVs, which is 5-fold higher than that of traditional ultracentrifugation. The sensitive detection of artificial EVs and internal miR-1290 was achieved with the limit of detection of 10 particles/μL and 0.07 amol, respectively. After lyophilization, CLHN-CCC shows no obvious loss of performance within 6 months, making it much more robust and user friendly. This technique could sensitively (sensitivity = 92.9%) and selectively (selectivity = 85.7%) identify low amount miR-1290 in serum EVs, distinguishing early-stage pancreatic cancer patients from healthy subjects, showing high potential for clinical applications.

RevDate: 2023-12-04
CmpDate: 2023-12-04

Canarutto D, Asperti C, Vavassori V, et al (2023)

Unbiased assessment of genome integrity and purging of adverse outcomes at the target locus upon editing of CD4[+] T-cells for the treatment of Hyper IgM1.

The EMBO journal, 42(23):e114188.

Hyper IgM1 is an X-linked combined immunodeficiency caused by CD40LG mutations, potentially treatable with CD4[+] T-cell gene editing with Cas9 and a "one-size-fits-most" corrective template. Contrary to established gene therapies, there is limited data on the genomic alterations following long-range gene editing, and no consensus on the relevant assays. We developed drop-off digital PCR assays for unbiased detection of large on-target deletions and found them at high frequency upon editing. Large deletions were also common upon editing different loci and cell types and using alternative Cas9 and template delivery methods. In CD40LG edited T cells, on-target deletions were counter-selected in culture and further purged by enrichment for edited cells using a selector coupled to gene correction. We then validated the sensitivity of optical genome mapping for unbiased detection of genome wide rearrangements and uncovered on-target trapping of one or more vector copies, which do not compromise functionality, upon editing using an integrase defective lentiviral donor template. No other recurring events were detected. Edited patient cells showed faithful reconstitution of CD40LG regulated expression and function with a satisfactory safety profile. Large deletions and donor template integrations should be anticipated and accounted for when designing and testing similar gene editing strategies.

RevDate: 2023-12-04
CmpDate: 2023-12-04

Wang J, Lin J, Chen Y, et al (2023)

An ultra-compact promoter drives widespread neuronal expression in mouse and monkey brains.

Cell reports, 42(11):113348.

Promoters are essential tools for basic and translational neuroscience research. An ideal promoter should possess the shortest possible DNA sequence with cell-type selectivity. However, whether ultra-compact promoters can offer neuron-specific expression is unclear. Here, we report the development of an extremely short promoter that enables selective gene expression in neurons, but not glial cells, in the brain. The promoter sequence originates from the human CALM1 gene and is only 120 bp in size. The CALM1 promoter (pCALM1) embedded in an adeno-associated virus (AAV) genome directed broad reporter expression in excitatory and inhibitory neurons in mouse and monkey brains. Moreover, pCALM1, when inserted into an all-in-one AAV vector expressing SpCas9 and sgRNA, drives constitutive and conditional in vivo gene editing in neurons and elicits functional alterations. These data demonstrate the ability of pCALM1 to conduct restricted neuronal gene expression, illustrating the feasibility of ultra-miniature promoters for targeting brain-cell subtypes.

RevDate: 2023-12-04
CmpDate: 2023-12-04

Qiu H, Li G, Yuan J, et al (2023)

Efficient exon skipping by base-editor-mediated abrogation of exonic splicing enhancers.

Cell reports, 42(11):113340.

Duchenne muscular dystrophy (DMD) is a severe genetic disease caused by the loss of the dystrophin protein. Exon skipping is a promising strategy to treat DMD by restoring truncated dystrophin. Here, we demonstrate that base editors (e.g., targeted AID-mediated mutagenesis [TAM]) are able to efficiently induce exon skipping by disrupting functional redundant exonic splicing enhancers (ESEs). By developing an unbiased and high-throughput screening to interrogate exonic sequences, we successfully identify novel ESEs in DMD exons 51 and 53. TAM-CBE (cytidine base editor) induces near-complete skipping of the respective exons by targeting these ESEs in patients' induced pluripotent stem cell (iPSC)-derived cardiomyocytes. Combined with strategies to disrupt splice sites, we identify suitable single guide RNAs (sgRNAs) with TAM-CBE to efficiently skip most DMD hotspot exons without substantial double-stranded breaks. Our study thus expands the repertoire of potential targets for CBE-mediated exon skipping in treating DMD and other RNA mis-splicing diseases.

RevDate: 2023-12-04
CmpDate: 2023-12-04

Liu J, Chen Y, Nong B, et al (2023)

CRISPR-assisted transcription activation by phase-separation proteins.

Protein & cell, 14(12):874-887.

The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 system has been widely used for genome engineering and transcriptional regulation in many different organisms. Current CRISPR-activation (CRISPRa) platforms often require multiple components because of inefficient transcriptional activation. Here, we fused different phase-separation proteins to dCas9-VPR (dCas9-VP64-P65-RTA) and observed robust increases in transcriptional activation efficiency. Notably, human NUP98 (nucleoporin 98) and FUS (fused in sarcoma) IDR domains were best at enhancing dCas9-VPR activity, with dCas9-VPR-FUS IDR (VPRF) outperforming the other CRISPRa systems tested in this study in both activation efficiency and system simplicity. dCas9-VPRF overcomes the target strand bias and widens gRNA designing windows without affecting the off-target effect of dCas9-VPR. These findings demonstrate the feasibility of using phase-separation proteins to assist in the regulation of gene expression and support the broad appeal of the dCas9-VPRF system in basic and clinical applications.

RevDate: 2023-12-01
CmpDate: 2023-12-01

Hao W, Chen Z, Tang J, et al (2023)

hnRNPA2B1 promotes the occurrence and progression of hepatocellular carcinoma by downregulating PCK1 mRNA via a m6A RNA methylation manner.

Journal of translational medicine, 21(1):861.

BACKGROUND: N6-methyladenosine (m6A) is the most prevalent RNA modification. Although hnRNPA2B1, as a reader of m6A modification, has been reported to promote tumorigenesis in a few types of tumors, its role in hepatocellular carcinoma (HCC) and the underlying molecular mechanism remains unclear.

METHODS: Multiple public databases were used to analyze the expression of hnRNPA2B1 in HCC and its correlation with survival prognosis. We employed a CRISPR-Cas9 sgRNA editing strategy to knockout hnRNPA2B1 expression in HCC cells. The biological function of hnRNPA2B1 in vitro in HCC cells was measured by CCK8, colony formation, migration, and invasion assay. The tumorigenic function of hnRNPA2B1 in vivo was determined by a subcutaneous tumor formation experiment and a HCC mouse model via tail injection of several plasmids into the mouse within 5s-7s. RNA binding protein immunoprecipitation (RIP) experiment using hnRNPA2B1 was performed to test the target genes of hnRNPA2B1 and methylated RNA immunoprecipitation (MeRIP) assay was performed to explore the m6A methylated mRNA of target genes.

RESULTS: hnRNPA2B1 highly expressed in HCC tissues, correlated with high grades and poor prognosis. Its knockout reduced HCC cell proliferation, migration, and invasion in vitro, while overexpression promoted these processes. hnRNPA2B1-knockout cells inhibited tumor formation in graft experiments. In HCC mice, endogenous knockout attenuated hepatocarcinogenesis. RNA-seq showed downregulated gluconeogenesis with high hnRNPA2B1 expression. hnRNPA2B1 negatively correlated with PCK1, a key enzyme. RIP assay revealed hnRNPA2B1 binding to PCK1 mRNA. hnRNPA2B1 knockout increased m6A-methylation of PCK1 mRNA. Interestingly, PCK1 knockout partially counteracted tumor inhibition by hnRNPA2B1 knockout in mice.

CONCLUSION: Our study indicated that hnRNPA2B1 is highly expressed in HCC and correlated with a poor prognosis. hnRNPA2B1 promotes the tumorigenesis and progression of HCC both in vitro and in vivo. Moreover, hnRNPA2B1 downregulates the expression of PCK1 mRNA via a m6A methylation manner. More importantly, the ability of hnRNPA2B1 to induce tumorigenesis and progression in HCC is dependent on its ability to decrease the expression of PCK1. Therefore, this study suggested that hnRNPA2B1 might be a diagnostic marker of poor prognosis of HCC and a potential therapeutic target for HCC patients.

RevDate: 2023-12-01
CmpDate: 2023-11-30

Yang Y, Mei H, Han X, et al (2023)

Synthetic CRISPR/dCas9-KRAB system driven by specific PSA promoter suppresses malignant biological behavior of prostate cancer cells through negative feedback inhibition of PSA expression.

Cellular & molecular biology letters, 28(1):96.

PSA is a type of proto-oncogene that is specifically and highly expressed in embryonic and prostate cancer cells, but not expressed in normal prostate tissue cells. The specific expression of prostate-specific antigen (PSA) is found to be related with the conditional transcriptional regulation of its promoter. Clustered regularly interspaced short palindromic repeats (CRISPR)-dCas9-KRAB is a newly developed transcriptional regulatory system that inhibits gene expression by interupting the DNA transcription process. Induction of CRISPR-dCas9-KRAB expression through the PSA promoter may help feedback inhibition of cellular PSA gene expression via single guide RNA (sgRNA), thereby monitoring and suppressing the malignant state of tumor cells. In this study, we examined the transcriptional activity of the PSA promoter in different prostate cancer cells and normal prostate epithelial cells and determined that it is indeed a prostate cancer cell-specific promoter.Then we constructed the CRISPR-dCas9-KRAB system driven by the PSA promoter, which can inhibit PSA gene expression in the prostate cancer cells at the transcriptional level, and therefore supress the malignant growth and migration of prostate cancer cells and promote their apoptosis in vitro. This study provides a potentially effective anti-cancer strategy for gene therapy of prostate cancer.

RevDate: 2023-11-28

Tan J, Shen M, Chai N, et al (2023)

Genome editing for plant synthetic metabolic engineering and developmental regulation.

Journal of plant physiology, 291:154141 pii:S0176-1617(23)00235-3 [Epub ahead of print].

Plant metabolism and development are a reflection of the orderly expression of genetic information intertwined with the environment interactions. Genome editing is the cornerstone for scientists to modify endogenous genes or introduce exogenous functional genes and metabolic pathways, holding immense potential applications in molecular breeding and biosynthesis. Over the course of nearly a decade of development, genome editing has advanced significantly beyond the simple cutting of double-stranded DNA, now enabling precise base and fragment replacements, regulation of gene expression and translation, as well as epigenetic modifications. However, the utilization of genome editing in plant synthetic metabolic engineering and developmental regulation remains exploratory. Here, we provide an introduction and a comprehensive overview of the editing attributes associated with various CRISPR/Cas tools, along with diverse strategies for the meticulous control of plant metabolic pathways and developments. Furthermore, we discuss the limitations of current approaches and future prospects for genome editing-driven plant breeding.

RevDate: 2023-11-28

Wimmer F, Englert F, Wandera KG, et al (2023)

Interrogating two extensively self-targeting Type I CRISPR-Cas systems in Xanthomonas albilineans reveals distinct anti-CRISPR proteins that block DNA degradation.

Nucleic acids research pii:7453255 [Epub ahead of print].

CRISPR-Cas systems store fragments of invader DNA as spacers to recognize and clear those same invaders in the future. Spacers can also be acquired from the host's genomic DNA, leading to lethal self-targeting. While self-targeting can be circumvented through different mechanisms, natural examples remain poorly explored. Here, we investigate extensive self-targeting by two CRISPR-Cas systems encoding 24 self-targeting spacers in the plant pathogen Xanthomonas albilineans. We show that the native I-C and I-F1 systems are actively expressed and that CRISPR RNAs are properly processed. When expressed in Escherichia coli, each Cascade complex binds its PAM-flanked DNA target to block transcription, while the addition of Cas3 paired with genome targeting induces cell killing. While exploring how X. albilineans survives self-targeting, we predicted putative anti-CRISPR proteins (Acrs) encoded within the bacterium's genome. Screening of identified candidates with cell-free transcription-translation systems and in E. coli revealed two Acrs, which we named AcrIC11 and AcrIF12Xal, that inhibit the activity of Cas3 but not Cascade of the respective system. While AcrF12Xal is homologous to AcrIF12, AcrIC11 shares sequence and structural homology with the anti-restriction protein KlcA. These findings help explain tolerance of self-targeting through two CRISPR-Cas systems and expand the known suite of DNA degradation-inhibiting Acrs.

RevDate: 2023-12-01
CmpDate: 2023-12-01

Fadul SM, Arshad A, R Mehmood (2023)

CRISPR-based epigenome editing: mechanisms and applications.

Epigenomics, 15(21):1137-1155.

Epigenomic anomalies contribute significantly to the development of numerous human disorders. The development of epigenetic research tools is essential for understanding how epigenetic marks contribute to gene expression. A gene-editing technique known as CRISPR (clustered regularly interspaced short palindromic repeats) typically targets a particular DNA sequence using a guide RNA (gRNA). CRISPR/Cas9 technology has been remodeled for epigenome editing by generating a 'dead' Cas9 protein (dCas9) that lacks nuclease activity and juxtaposing it with an epigenetic effector domain. Based on fusion partners of dCas9, a specific epigenetic state can be achieved. CRISPR-based epigenome editing has widespread application in drug screening, cancer treatment and regenerative medicine. This paper discusses the tools developed for CRISPR-based epigenome editing and their applications.

RevDate: 2023-11-29

Deol P, Madhwal A, Sharma G, et al (2022)

CRISPR use in diagnosis and therapy for COVID-19.

Methods in microbiology, 50:123-150.

Since the beginning of the COVID-19 pandemic, many diagnostic approaches (RT-qPCR, RAPID, LFA) have been adopted, with RT-qPCR being the most popular/gold standard. But, one of the major problems of COVID-19 diagnostics is the presentation of a wide range of symptoms which varies among different patients and needs early diagnosis for better management. Even though RT-qPCR is a precise molecular technique false negative results may be obtained. On the other hand, CRISPR-based SARS-CoV-2 detection approaches are cost and time efficient, highly sensitive and specific, and do not require sophisticated instruments. Moreover, they also show promise for increased scalability and diagnostic tests can be carried out at the point-of-care (POC). The CRISPR can be customized to the target of any genomic region of interest within the desired genome possessing a broad range of other applications and has been efficiently implemented for diagnosis of SARS-CoV-2. The CRISPR/Cas systems provide the specific gene targeting with immense potential to develop new generation diagnostics and therapeutics. Moreover, with the CRISPR/Cas based therapeutics, multiplexing is possible, where different sgRNAs or crRNAs can be guided to more than one target within the same gene thus decreasing the possibility of viral escape mutants. As an exceptionally efficient tool CRISPR/Cas13 and CARVER (Cas13-assisted restriction of viral expression and readout) systems can be implemented to target a broad range of ssRNA viruses that can be used for both, diagnosis and treatment for a variety of viral diseases including SARS-CoV-2. However, the efficacy and safety of the CRISPR-based therapeutics needs to be assessed in pre-clinical and clinical settings. Although the CRISPR biotechnologies are not very helpful to control the present pandemic of COVID-19 it is hopeful that the limitations of the CRISPR/Cas system can be overcome in the near future. The CRISPR based strategies may lead to a new era in the field of disease diagnosis and therapeutic development that would make us better prepared for future viral threats.

RevDate: 2023-11-30
CmpDate: 2023-11-29

Javadi M, Sazegar H, A Doosti (2023)

Genome editing approaches with CRISPR/Cas9: the association of NOX4 expression in breast cancer patients and effectiveness evaluation of different strategies of CRISPR/Cas9 to knockout Nox4 in cancer cells.

BMC cancer, 23(1):1155.

BACKGROUND: The increasing prevalence of cancer detection necessitated practical strategies to deliver highly accurate, beneficial, and dependable processed information together with experimental results. We deleted the cancer biomarker NOX4 using three novel genetic knockout (KO) methods. Homology-directed repair (HDR), Dual allele HITI (Du-HITI) and CRISPR-excision were utilized in this study.

METHODS: The predictive value of the NOX4 expression profile was assessed using a combined hazard ratio (HR) with a 95% confidence interval (CI). With a 95% confidence interval, a pooled odd ratio (OR) was used to calculate the relationship between NOX4 expression patterns and cancer metastasis. There were 1060 tumor patients in all sixteen research that made up this meta-analysis. To stop the NOX4 from being transcribed, we employed three different CRISPR/Cas9-mediated knockdown methods. The expression of RNA was assessed using RT-PCR. We employed the CCK-8 assay, colony formation assays, and the invasion transwell test for our experiments measuring cell proliferation and invasion. Using a sphere-formation test, the stemness was determined. Luciferase reporter tests were carried out to verify molecular adhesion. Utilizing RT-qPCR, MTT, and a colony formation assay, the functional effects of NOX4 genetic mutation in CRISPR-excision, CRISPR-HDR, and CRISPR du-HITI knockdown cell lines of breast cancer were verified.

RESULTS: There were 1060 malignant tumors in the 16 studies that made up this meta-analysis. In the meta-analysis, higher NOX4 expression was linked to both a shorter overall survival rate (HR = 1.93, 95% CI 1.49-2.49, P < 0.001) and a higher percentage of lymph node metastases (OR = 3.22, 95% CI 2.18-4.29, P < 0.001). In breast carcinoma cells, it was discovered that NOX4 was overexpressed, and this increase was linked to a poor prognosis. The gain and loss-of-function assays showed enhanced NOX4 breast carcinoma cell proliferation, sphere-forming capacity, and tumor development. To activate transcription, the transcriptional factor E2F1 also attaches to the promoter region of the Nanog gene. The treatment group (NOX4 ablation) had substantially more significant levels of proapoptotic gene expression than the control group (P < 0.01). Additionally, compared to control cells, mutant cells expressed fewer antiapoptotic genes (P < 0.001). The du-HITI technique incorporated a reporter and a transcription termination marker into the two target alleles. Both donor vector preparation and cell selection were substantially simpler using this approach than with "CRISPR HDR" or "CRISPR excision." Furthermore, single-cell knockouts for both genotypes were created when this method was applied in the initial transfection experiment.

CONCLUSIONS: The NOX4 Knockout cell lines generated in this research may be used for additional analytical studies to reveal the entire spectrum of NOX4 activities. The du-HITI method described in this study was easy to employ and could produce homozygous individuals who were knockout for a specific protein of interest.

RevDate: 2023-11-30
CmpDate: 2023-11-29

Nan X, Hardinge P, Hoehn S, et al (2023)

VarLOCK: sequencing-independent, rapid detection of SARS-CoV-2 variants of concern for point-of-care testing, qPCR pipelines and national wastewater surveillance.

Scientific reports, 13(1):20832.

The COVID-19 pandemic demonstrated the need for rapid molecular diagnostics. Vaccination programs can provide protection and facilitate the opening of society, but newly emergent and existing viral variants capable of evading the immune system endanger their efficacy. Effective surveillance for Variants of Concern (VOC) is therefore important. Rapid and specific molecular diagnostics can provide speed and coverage advantages compared to genomic sequencing alone, benefitting the public health response and facilitating VOC containment. Here we expand the recently developed SARS-CoV-2 CRISPR-Cas detection technology (SHERLOCK) to provide rapid and sensitive discrimination of SARS-CoV-2 VOCs that can be used at point of care, implemented in the pipelines of small or large testing facilities, and even determine the proportion of VOCs in pooled population-level wastewater samples. This technology complements sequencing efforts to allow facile and rapid identification of individuals infected with VOCs to help break infection chains. We show the optimisation of our VarLOCK assays (Variant-specific SHERLOCK) for multiple specific mutations in the S gene of SARS-CoV-2 and validation with samples from the Cardiff University Testing Service. We also show the applicability of VarLOCK to national wastewater surveillance of SARS-CoV-2 variants and the rapid adaptability of the technique for new and emerging VOCs.

RevDate: 2023-11-27

Tripathi JN, Ntui VO, L Tripathi (2023)

Precision genetics tools for genetic improvement of banana.

The plant genome [Epub ahead of print].

Banana is an important food security crop for millions of people in the tropics but it faces challenges from diseases and pests. Traditional breeding methods have limitations, prompting the exploration of precision genetic tools like genetic modification and genome editing. Extensive efforts using transgenic approaches have been made to develop improved banana varieties with resistance to banana Xanthomonas wilt, Fusarium wilt, and nematodes. However, these efforts should be extended for other pests, diseases, and abiotic stresses. The commercialization of transgenic crops still faces continuous challenges with regulatory and public acceptance. Genome editing, particularly CRISPR/Cas, offers precise modifications to the banana genome and has been successfully applied in the improvement of banana. Targeting specific genes can contribute to the development of improved banana varieties with enhanced resistance to various biotic and abiotic constraints. This review discusses recent advances in banana improvement achieved through genetic modification and genome editing.

RevDate: 2023-11-27

He Y, Shao S, J Chen (2023)

High-Fidelity Identification of Single Nucleotide Polymorphism by Type V CRISPR Systems.

ACS sensors [Epub ahead of print].

Accurate and sensitive detection of single nucleotide polymorphism (SNP) holds significant clinical implications, especially in the field of cancer diagnosis. Leveraging its high accuracy and programmability, the CRISPR system emerges as a promising platform for advancing the identification of SNPs. In this study, we compared two type V CRISPR/Cas systems (Cas12a and Cas14a) for the identification of cancer-related SNP. Their identification performances were evaluated by characterizing their mismatch tolerance to the BRAF gene. We found that the CRISPR/Cas14a system exhibited superior accuracy and robustness over the CRISPR/Cas12a system for SNP detection. Furthermore, blocker displacement amplification (BDA) was combined with the CRISPR/Cas14a system to eliminate the interference of the wild type (WT) and increase the detection accuracy. In this strategy, we were able to detect BRAF V600E as low as 10[3] copies with a sensitivity of 0.1% variant allele frequency. Moreover, the BDA-assisted CRISPR/Cas14a system has been applied to identify the BRAF mutation from human colorectal carcinoma cells, achieving a high sensitivity of 0.5% variant allele frequency, which is comparable to or even superior to those of most commercially available products. This work has broadened the scope of the CRISPR system and provided a promising method for precision medicine.

RevDate: 2023-11-29
CmpDate: 2023-11-29

Thomsen MK (2023)

The double-edge sword of CRISPR application for in vivo studies.

Oncotarget, 14:919-920.

RevDate: 2023-11-27

Liu J, Jaffe AL, Chen L, et al (2023)

Host translation machinery is not a barrier to phages that interact with both CPR and non-CPR bacteria.

mBio [Epub ahead of print].

Here, we profiled putative phages of Saccharibacteria, which are of particular importance as Saccharibacteria influence some human oral diseases. We additionally profiled putative phages of Gracilibacteria and Absconditabacteria, two Candidate Phyla Radiation (CPR) lineages of interest given their use of an alternative genetic code. Among the phages identified in this study, some are targeted by spacers from both CPR and non-CPR bacteria and others by both bacteria that use the standard genetic code as well as bacteria that use an alternative genetic code. These findings represent new insights into possible phage replication strategies and have relevance for phage therapies that seek to manipulate microbiomes containing CPR bacteria.

RevDate: 2023-11-27

Muzyukina P, Shkaruta A, Guzman NM, et al (2023)

Identification of an anti-CRISPR protein that inhibits the CRISPR-Cas type I-B system in Clostridioides difficile.

mSphere [Epub ahead of print].

Clostridioides difficile is the widespread anaerobic spore-forming bacterium that is a major cause of potentially lethal nosocomial infections associated with antibiotic therapy worldwide. Due to the increase in severe forms associated with a strong inflammatory response and higher recurrence rates, a current imperative is to develop synergistic and alternative treatments for C. difficile infections. In particular, phage therapy is regarded as a potential substitute for existing antimicrobial treatments. However, it faces challenges because C. difficile has highly active CRISPR-Cas immunity, which may be a specific adaptation to phage-rich and highly crowded gut environment. To overcome this defense, C. difficile phages must employ anti-CRISPR mechanisms. Here, we present the first anti-CRISPR protein that inhibits the CRISPR-Cas defense system in this pathogen. Our work offers insights into the interactions between C. difficile and its phages, paving the way for future CRISPR-based applications and development of effective phage therapy strategies combined with the engineering of virulent C. difficile infecting phages.

RevDate: 2023-11-26

Wang Q, Ren Y, Meng T, et al (2023)

Cas14a1-advanced LAMP for ultrasensitive and visual Pathogen diagnostic.

Talanta, 269:125458 pii:S0039-9140(23)01209-2 [Epub ahead of print].

Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas enzymes have been widely applied for biosensor development, combined with various isothermal amplification strategies (IAS) to boost sensitivity and specificity. Currently, the unstable assay and tedious manipulation usually hinder its practical applications. Here, a Cas14a1-advanced LAMP assay (CALA) combined with Rapid Extraction of Bacterial Genomic DNA (REBGD) is proposed for pathogen detection. For rapid CALA, a single stranded fluorescence reporter and ssDNA-gold nanoparticles (AuNPs) are used as signal indicators to establish ultrasensitive and visual platforms. This assay displays precise detection of bacteria, which can achieve an ultrasensitive limit of detection (LOD) 10 aM target genomic DNA. Furthermore, the high reliability of pathogen diagnostic for contrived samples is validated through the rapid visual CALA platform, demonstrating the promising practical testing availability of pathogen detection.

RevDate: 2023-11-28
CmpDate: 2023-11-28

Feng Q, J Li (2023)

Construction and Validation of a CRISPR/dCpf1-Based Transcriptional Regulatory System in Saccharomyces Cerevisiae.

Studies in health technology and informatics, 308:680-688.

Saccharomyces cerevisiae has been extensively studied and applied as a model microbial platform for efficient and sustainable production of biofuels, chemicals and natural products. In recent years, the application of CRISPR system in genome editing and transcriptional regulation has greatly improved the efficiency of microbial genome editing. To achieve transcriptional regulation the CRISPR/dCpf1-mediated CRISPRa/i transcriptional regulation system was constructed in this study. By constructing crRNA arrays and adding different activation proteins, repression efficiencies from 62% to 177% for the CRISPRi system and activation efficiencies from 154% to 320% for the CRISPRa system were achieved in the eGFP gene reporter system.

RevDate: 2023-11-27
CmpDate: 2023-11-27

Chung T, Merrill JR, SK Lyons (2024)

CRISPR/Cas for PET Reporter Gene Engineering.

Methods in molecular biology (Clifton, N.J.), 2729:285-301.

The relatively recent discovery of CRISPR/Cas has led to a revolution in our ability to efficiently manipulate the genome of eukaryotic cells. We describe here a protocol that employs CRISPR technology to precisely knock-in a PET imaging reporter transgene into a specific genetic locus of interest. Resulting transcription of the targeted reporter will more accurately mimic physiologic expression of the endogenous allele than conventional approaches, and so this method has the potential to become an efficient way to generate a new generation of "gold-standard" reporter transgenes. We break down the protocol into three experimental stages: how to identify the genomic location that the reporter transgene will be inserted, how to practically insert the reporter transgene into the genome, and how to screen resultant clones for the correct targeted event.

RevDate: 2023-11-30
CmpDate: 2023-11-30

Cao H, Mao K, Zhang H, et al (2024)

Thermal stability and micrdose-based coupling CRISPR/Cas12a biosensor for amplification-free detection of hgcA gene in paddy soil.

The Science of the total environment, 909:168536.

The lack of point-of-use (POU) methods hinders the utilization of the hgcA gene to rapidly evaluate methylmercury risks. CRISPR/Cas12a is a promising technology, but shortcomings such as low sensitivity, a strict reaction temperature and high background signal limit its further utilization. Here, a thermally stable microsystem-based CRISPR/Cas12a biosensor was constructed to achieve POU analysis for hgcA. First, three target gRNAs were designed to recognize hgcA. Then, a microsystem was developed to eliminate the background signal. Next, the effect of temperature on the activity of the Cas12a-gRNA complex was explored and its thermal stability was discovered. After that, coupling gRNA assay was introduced to improve sensitivity, exhibiting a limit of detection as low as 0.49 pM with a linear range of 0.98-125 pM, and a recovery rate between 90 and 110 % for hgcA. The biosensor was finally utilized to assess hgcA abundance in paddy soil, and high abundance of hgcA was found in these paddy soil samples. This study not only systematically explored the influence of temperature and microsystem on CRISPR/Cas12a, providing vital references for other novel CRISPR-based detection methods, but also applied the CRISPR-based analytical method to the field of environmental geochemistry for the first time, demonstrating enormous potential for POU detection in this field.

RevDate: 2023-11-30
CmpDate: 2023-11-30

Sasaki-Honda M, Akatsuka K, T Sawai (2023)

Is epigenome editing non-inheritable? Implications for ethics and the regulation of human applications.

Stem cell reports, 18(11):2005-2009.

Epigenome editing offers ethical advantages with non-inheritable gene expression control. However, concerns arise regarding potential transgenerational effects in humans. Ethical and regulatory evaluation is crucial, considering recent advancements and enhanced understanding of transgenerational epigenetics in both mammals and humans.

RevDate: 2023-11-30
CmpDate: 2023-11-30

Crunkhorn S (2023)

Engineering a compact genome-editing tool.

Nature reviews. Drug discovery, 22(12):953.

RevDate: 2023-11-30
CmpDate: 2023-04-25

Diaz Quiroz JF, Ojha N, Shayhidin EE, et al (2023)

Development of a selection assay for small guide RNAs that drive efficient site-directed RNA editing.

Nucleic acids research, 51(7):e41.

A major challenge confronting the clinical application of site-directed RNA editing (SDRE) is the design of small guide RNAs (gRNAs) that can drive efficient editing. Although many gRNA designs have effectively recruited endogenous Adenosine Deaminases that Act on RNA (ADARs), most of them exceed the size of currently FDA-approved antisense oligos. We developed an unbiased in vitro selection assay to identify short gRNAs that promote superior RNA editing of a premature termination codon. The selection assay relies on hairpin substrates in which the target sequence is linked to partially randomized gRNAs in the same molecule, so that gRNA sequences that promote editing can be identified by sequencing. These RNA substrates were incubated in vitro with ADAR2 and the edited products were selected using amplification refractory mutation system PCR and used to regenerate the substrates for a new round of selection. After nine repetitions, hairpins which drove superior editing were identified. When gRNAs of these hairpins were delivered in trans, eight of the top ten short gRNAs drove superior editing both in vitro and in cellula. These results show that efficient small gRNAs can be selected using our approach, an important advancement for the clinical application of SDRE.

RevDate: 2023-11-30
CmpDate: 2022-12-19

Wang Y, Cottle WT, Wang H, et al (2022)

Achieving single nucleotide sensitivity in direct hybridization genome imaging.

Nature communications, 13(1):7776.

Direct visualization of point mutations in situ can be informative for studying genetic diseases and nuclear biology. We describe a direct hybridization genome imaging method with single-nucleotide sensitivity, single guide genome oligopaint via local denaturation fluorescence in situ hybridization (sgGOLDFISH), which leverages the high cleavage specificity of eSpCas9(1.1) variant combined with a rationally designed guide RNA to load a superhelicase and reveal probe binding sites through local denaturation. The guide RNA carries an intentionally introduced mismatch so that while wild-type target DNA sequence can be efficiently cleaved, a mutant sequence with an additional mismatch (e.g., caused by a point mutation) cannot be cleaved. Because sgGOLDFISH relies on genomic DNA being cleaved by Cas9 to reveal probe binding sites, the probes will only label the wild-type sequence but not the mutant sequence. Therefore, sgGOLDFISH has the sensitivity to differentiate the wild-type and mutant sequences differing by only a single base pair. Using sgGOLDFISH, we identify base-editor-modified and unmodified progeroid fibroblasts from a heterogeneous population, validate the identification through progerin immunofluorescence, and demonstrate accurate sub-nuclear localization of point mutations.

RevDate: 2023-11-27
CmpDate: 2023-11-27

Kang KK, YG Cho (2023)

Genetic Analysis Based on CRISPR/Cas9 Technology in Plants.

International journal of molecular sciences, 24(22):.

Genome-editing technology is a type of genetic engineering in which DNA is inserted into, replaced in, or deleted from the genome using artificially engineered nucleases or genetic scissors [...].

RevDate: 2023-11-27
CmpDate: 2023-11-27

Ding S, Liu J, Han X, et al (2023)

CRISPR/Cas9-Mediated Genome Editing in Cancer Therapy.

International journal of molecular sciences, 24(22):.

The Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein 9 (CRISPR/Cas9) system, an RNA-based adaptive immune system found in bacteria and archaea, has catalyzed the development and application of a new generation of gene editing tools. Numerous studies have shown that this system can precisely target a wide range of human genes, including those associated with diseases such as cancer. In cancer research, the intricate genetic mutations in tumors have promoted extensive utilization of the CRISPR/Cas9 system due to its efficient and accurate gene editing capabilities. This includes improvements in Chimeric Antigen Receptor (CAR)-T-cell therapy, the establishment of tumor models, and gene and drug target screening. Such progress has propelled the investigation of cancer molecular mechanisms and the advancement of precision medicine. However, the therapeutic potential of genome editing remains underexplored, and lingering challenges could elevate the risk of additional genetic mutations. Here, we elucidate the fundamental principles of CRISPR/Cas9 gene editing and its practical applications in tumor research. We also briefly discuss the primary challenges faced by CRISPR technology and existing solutions, intending to enhance the efficacy of this gene editing therapy and shed light on the underlying mechanisms of tumors.

RevDate: 2023-11-27
CmpDate: 2023-11-27

Movahedi A, Aghaei-Dargiri S, Li H, et al (2023)

CRISPR Variants for Gene Editing in Plants: Biosafety Risks and Future Directions.

International journal of molecular sciences, 24(22):.

The CRISPR genome editing technology is a crucial tool for enabling revolutionary advancements in plant genetic improvement. This review shows the latest developments in CRISPR/Cas9 genome editing system variants, discussing their benefits and limitations for plant improvement. While this technology presents immense opportunities for plant breeding, it also raises serious biosafety concerns that require careful consideration, including potential off-target effects and the unintended transfer of modified genes to other organisms. This paper highlights strategies to mitigate biosafety risks and explores innovative plant gene editing detection methods. Our review investigates the international biosafety guidelines for gene-edited crops, analyzing their broad implications for agricultural and biotechnology research and advancement. We hope to provide illuminating and refined perspectives for industry practitioners and policymakers by evaluating CRISPR genome enhancement in plants.

RevDate: 2023-11-27
CmpDate: 2023-11-27

Davis-Anderson K, Micheva-Viteva S, Solomon E, et al (2023)

CRISPR/Cas9 Directed Reprogramming of iPSC for Accelerated Motor Neuron Differentiation Leads to Dysregulation of Neuronal Fate Patterning and Function.

International journal of molecular sciences, 24(22):.

Neurodegeneration causes a significant disease burden and there are few therapeutic interventions available for reversing or slowing the disease progression. Induced pluripotent stem cells (iPSCs) hold significant potential since they are sourced from adult tissue and have the capacity to be differentiated into numerous cell lineages, including motor neurons. This differentiation process traditionally relies on cell lineage patterning factors to be supplied in the differentiation media. Genetic engineering of iPSC with the introduction of recombinant master regulators of motor neuron (MN) differentiation has the potential to shorten and streamline cell developmental programs. We have established stable iPSC cell lines with transient induction of exogenous LHX3 and ISL1 from the Tet-activator regulatory region and have demonstrated that induction of the transgenes is not sufficient for the development of mature MNs in the absence of neuron patterning factors. Comparative global transcriptome analysis of MN development from native and Lhx-ISL1 modified iPSC cultures demonstrated that the genetic manipulation helped to streamline the neuronal patterning process. However, leaky gene expression of the exogenous MN master regulators in iPSC resulted in the premature activation of genetic pathways characteristic of the mature MN function. Dysregulation of metabolic and regulatory pathways within the developmental process affected the MN electrophysiological responses.

RevDate: 2023-11-27
CmpDate: 2023-11-27

Nagy B, Öktem A, Ferenc G, et al (2023)

CRISPR/Cas9 Mutagenesis through Introducing a Nanoparticle Complex Made of a Cationic Polymer and Nucleic Acids into Maize Protoplasts.

International journal of molecular sciences, 24(22):.

Presently, targeted gene mutagenesis attracts increasing attention both in plant research and crop improvement. In these approaches, successes are largely dependent on the efficiency of the delivery of gene editing components into plant cells. Here, we report the optimization of the cationic polymer poly(2-hydroxypropylene imine) (PHPI)-mediated delivery of plasmid DNAs, or single-stranded oligonucleotides labelled with Cyanine3 (Cy3) or 6-Carboxyfluorescein (6-FAM)-fluorescent dyes into maize protoplasts. Co-delivery of the GFP-expressing plasmid and the Cy3-conjugated oligonucleotides has resulted in the cytoplasmic and nuclear accumulation of the green fluorescent protein and a preferential nuclear localization of oligonucleotides. We show the application of nanoparticle complexes, i.e., "polyplexes" that comprise cationic polymers and nucleic acids, for CRISPR/Cas9 editing of maize cells. Knocking out the functional EGFP gene in transgenic maize protoplasts was achieved through the co-delivery of plasmids encoding components of the editing factors Cas9 (pFGC-pcoCas9) and gRNA (pZmU3-gRNA) after complexing with a cationic polymer (PHPI). Several edited microcalli were identified based on the lack of a GFP fluorescence signal. Multi-base and single-base deletions in the EGFP gene were confirmed using Sanger sequencing. The presented results support the use of the PHPI cationic polymer in plant protoplast-mediated genome editing approaches.

RevDate: 2023-11-29
CmpDate: 2023-11-29

Mao K, Zhang H, Ran F, et al (2024)

Portable biosensor combining CRISPR/Cas12a and loop-mediated isothermal amplification for antibiotic resistance gene ermB in wastewater.

Journal of hazardous materials, 462:132793.

Wastewater is among the main sources of antibiotic resistance genes (ARGs) in the environment, but effective methods to quickly assess ARGs on-site in wastewater are lacking. Here, using the typical ARG ermB as the target, we report a portable biosensor combining CRISPR/Cas12a and loop-mediated isothermal amplification (LAMP) for the detection of ARGs. Six primers of LAMP and the crRNA of CRISPR/Cas12a were first designed to be preamplification with LAMP and lead Cas12a to recognize the ermB via base pairing. Due to the trans-cleavage activity of CRISPR/Cas12a after amplicon recognition, ssDNA probes modified with reporter molecules were used to implement a visual assay with lateral flow test strips and fluorescence. After a simple nucleic acid extraction with magnetic beads, the constructed biosensor possesses excellent sensitivity and selectivity as low as 2.75 × 10[3] copies/μL using fluorescence and later flow strips in wastewater. We further evaluated the community-wide prevalence of ermB in wastewater influent and found high mass loads of ermB during different months. This user-friendly and low-cost biosensor is applicable for rapid on-site ARG detection, providing a potential point-of-use method for rapid assessments of ARG abundance in wastewater from large city areas with many wastewater treatment plants and in resource-limited rural areas.

RevDate: 2023-11-29
CmpDate: 2023-11-29

Tulkens D, Boelens M, Naert T, et al (2023)

Mutations in the histone methyltransferase Ezh2 drive context-dependent leukemia in Xenopus tropicalis.

Leukemia, 37(12):2404-2413.

CRISPR-mediated simultaneous targeting of candidate tumor suppressor genes in Xenopus tropicalis allows fast functional assessment of co-driver genes for various solid tumors. Genotyping of tumors that emerge in the mosaic mutant animals rapidly exposes the gene mutations under positive selection for tumor establishment. However, applying this simple approach to the blood lineage has not been attempted. Multiple hematologic malignancies have mutations in EZH2, encoding the catalytic subunit of the Polycomb Repressive Complex 2. Interestingly, EZH2 can act as an oncogene or a tumor suppressor, depending on cellular context and disease stage. We show here that mosaic CRISPR/Cas9 mediated ezh2 disruption in the blood lineage resulted in early and penetrant acute myeloid leukemia (AML) induction. While animals were co-targeted with an sgRNA that induces notch1 gain-of-function mutations, sequencing of leukemias revealed positive selection towards biallelic ezh2 mutations regardless of notch1 mutational status. Co-targeting dnm2, recurrently mutated in T/ETP-ALL, induced a switch from myeloid towards acute T-cell leukemia. Both myeloid and T-cell leukemias engrafted in immunocompromised hosts. These data underline the potential of Xenopus tropicalis for modeling human leukemia, where mosaic gene disruption, combined with deep amplicon sequencing of the targeted genomic regions, can rapidly and efficiently expose co-operating driver gene mutations.

RevDate: 2023-11-29
CmpDate: 2023-11-29

Raban R, Marshall JM, Hay BA, et al (2023)

Manipulating the Destiny of Wild Populations Using CRISPR.

Annual review of genetics, 57:361-390.

Genetic biocontrol aims to suppress or modify populations of species to protect public health, agriculture, and biodiversity. Advancements in genome engineering technologies have fueled a surge in research in this field, with one gene editing technology, CRISPR, leading the charge. This review focuses on the current state of CRISPR technologies for genetic biocontrol of pests and highlights the progress and ongoing challenges of using these approaches.

RevDate: 2023-11-27
CmpDate: 2023-11-27

Tyumentseva M, Tyumentsev A, V Akimkin (2023)

CRISPR/Cas9 Landscape: Current State and Future Perspectives.

International journal of molecular sciences, 24(22):.

CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 is a unique genome editing tool that can be easily used in a wide range of applications, including functional genomics, transcriptomics, epigenetics, biotechnology, plant engineering, livestock breeding, gene therapy, diagnostics, and so on. This review is focused on the current CRISPR/Cas9 landscape, e.g., on Cas9 variants with improved properties, on Cas9-derived and fusion proteins, on Cas9 delivery methods, on pre-existing immunity against CRISPR/Cas9 proteins, anti-CRISPR proteins, and their possible roles in CRISPR/Cas9 function improvement. Moreover, this review presents a detailed outline of CRISPR/Cas9-based diagnostics and therapeutic approaches. Finally, the review addresses the future expansion of genome editors' toolbox with Cas9 orthologs and other CRISPR/Cas proteins.

RevDate: 2023-11-27
CmpDate: 2023-11-27

Mohammadian Gol T, Kim M, Sinn R, et al (2023)

CRISPR-Cas9-Based Gene Knockout of Immune Checkpoints in Expanded NK Cells.

International journal of molecular sciences, 24(22):.

Natural killer (NK) cell immunotherapy has emerged as a novel treatment modality for various cancer types, including leukemia. The modulation of inhibitory signaling pathways in T cells and NK cells has been the subject of extensive investigation in both preclinical and clinical settings in recent years. Nonetheless, further research is imperative to optimize antileukemic activities, especially regarding NK-cell-based immunotherapies. The central scientific question of this study pertains to the potential for boosting cytotoxicity in expanded and activated NK cells through the inhibition of inhibitory receptors. To address this question, we employed the CRISPR-Cas9 system to target three distinct inhibitory signaling pathways in NK cells. Specifically, we examined the roles of A2AR within the metabolic purinergic signaling pathway, CBLB as an intracellular regulator in NK cells, and the surface receptors NKG2A and CD96 in enhancing the antileukemic efficacy of NK cells. Following the successful expansion of NK cells, they were transfected with Cas9+sgRNA RNP to knockout A2AR, CBLB, NKG2A, and CD96. The analysis of indel frequencies for all four targets revealed good knockout efficiencies in expanded NK cells, resulting in diminished protein expression as confirmed by flow cytometry and Western blot analysis. Our in vitro killing assays demonstrated that NKG2A and CBLB knockout led to only a marginal improvement in the cytotoxicity of NK cells against AML and B-ALL cells. Furthermore, the antileukemic activity of CD96 knockout NK cells did not yield significant enhancements, and the blockade of A2AR did not result in significant improvement in killing efficiency. In conclusion, our findings suggest that CRISPR-Cas9-based knockout strategies for immune checkpoints might not be sufficient to efficiently boost the antileukemic functions of expanded (and activated) NK cells and, at the same time, point to the need for strong cellular activating signals, as this can be achieved, for example, via transgenic chimeric antigen receptor expression.

RevDate: 2023-11-27
CmpDate: 2023-11-27

Güngör B, Biró JB, Domonkos Á, et al (2023)

Targeted mutagenesis of Medicago truncatula Nodule-specific Cysteine-Rich (NCR) genes using the Agrobacterium rhizogenes-mediated CRISPR/Cas9 system.

Scientific reports, 13(1):20676.

The host-produced nodule specific cysteine-rich (NCR) peptides control the terminal differentiation of endosymbiotic rhizobia in the nodules of IRLC legumes. Although the Medicago truncatula genome encodes about 700 NCR peptides, only few of them have been proven to be crucial for nitrogen-fixing symbiosis. In this study, we applied the CRISPR/Cas9 gene editing technology to generate knockout mutants of NCR genes for which no genetic or functional data were previously available. We have developed a workflow to analyse the mutation and the symbiotic phenotype of individual nodules formed on Agrobacterium rhizogenes-mediated transgenic hairy roots. The selected NCR genes were successfully edited by the CRISPR/Cas9 system and nodules formed on knockout hairy roots showed wild type phenotype indicating that peptides NCR068, NCR089, NCR128 and NCR161 are not essential for symbiosis between M. truncatula Jemalong and Sinorhizobium medicae WSM419. We regenerated stable mutants edited for the NCR068 from hairy roots obtained by A. rhizogenes-mediated transformation. The analysis of the symbiotic phenotype of stable ncr068 mutants showed that peptide NCR068 is not required for symbiosis with S. meliloti strains 2011 and FSM-MA either. Our study reports that gene editing can help to elicit the role of certain NCRs in symbiotic nitrogen fixation.

RevDate: 2023-11-28
CmpDate: 2023-11-28

Witkowsky L, Norstad M, Glynn AR, et al (2023)

Towards affordable CRISPR genomic therapies: a task force convened by the Innovative Genomics Institute.

Gene therapy, 30(10-11):747-752.

RevDate: 2023-11-27
CmpDate: 2023-11-27

Li Y, Wu Y, Xu R, et al (2023)

In vivo imaging of mitochondrial DNA mutations using an integrated nano Cas12a sensor.

Nature communications, 14(1):7722.

Mutations in mitochondrial DNA (mtDNA) play critical roles in many human diseases. In vivo visualization of cells bearing mtDNA mutations is important for resolving the complexity of these diseases, which remains challenging. Here we develop an integrated nano Cas12a sensor (InCasor) and show its utility for efficient imaging of mtDNA mutations in live cells and tumor-bearing mouse models. We co-deliver Cas12a/crRNA, fluorophore-quencher reporters and Mg[2+] into mitochondria. This process enables the activation of Cas12a's trans-cleavage by targeting mtDNA, which efficiently cleave reporters to generate fluorescent signals for robustly sensing and reporting single-nucleotide variations (SNVs) in cells. Since engineered crRNA significantly increase Cas12a's sensitivity to mismatches in mtDNA, we can identify tumor tissue and metastases by visualizing cells with mutant mtDNAs in vivo using InCasor. This CRISPR imaging nanoprobe holds potential for applications in mtDNA mutation-related basic research, diagnostics and gene therapies.

RevDate: 2023-11-27
CmpDate: 2023-11-27

Kim Y, HM Lee (2023)

CRISPR-Cas System Is an Effective Tool for Identifying Drug Combinations That Provide Synergistic Therapeutic Potential in Cancers.

Cells, 12(22):.

Despite numerous efforts, the therapeutic advancement for neuroblastoma and other cancer treatments is still ongoing due to multiple challenges, such as the increasing prevalence of cancers and therapy resistance development in tumors. To overcome such obstacles, drug combinations are one of the promising applications. However, identifying and implementing effective drug combinations are critical for achieving favorable treatment outcomes. Given the enormous possibilities of combinations, a rational approach is required to predict the impact of drug combinations. Thus, CRISPR-Cas-based and other approaches, such as high-throughput pharmacological and genetic screening approaches, have been used to identify possible drug combinations. In particular, the CRISPR-Cas system (Clustered Regularly Interspaced Short Palindromic Repeats) is a powerful tool that enables us to efficiently identify possible drug combinations that can improve treatment outcomes by reducing the total search space. In this review, we discuss the rational approaches to identifying, examining, and predicting drug combinations and their impact.

RevDate: 2023-11-27
CmpDate: 2023-11-27

Yun D, C Jung (2023)

MiRNA-Responsive CRISPR-Cas System via a DNA Regulator.

Biosensors, 13(11):.

Clustered regularly interspaced short palindromic repeats (CRISPR)- CRISPR-associated protein 9 (Cas9) genome editing technology is widely used for gene editing because it provides versatility in genetic manipulation. Several methods for regulating CRISPR activity already exist for accurate editing, but these require complex engineering. Thus, a simple and convenient regulatory system is required. In this study, we devised a CRISPR activation system using a DNA regulator that can be activated by miRNAs. The designed regulator was divided into two parts. The inhibition component consisted of the protospacer-adjacent motif (PAM) and seed sequence, which are important for Cas9 target recognition and bind to the ribonucleoprotein (RNP) complex for inhibition. The miRNA recognition component has a single-stranded toehold DNA for target miRNA binding and a partial double-stranded DNA complementary to the remaining miRNA sequence. In the presence of target miRNAs, the structure of the regulator is disrupted by the miRNAs, leading to its dissociation from the RNP complex and subsequent restoration of CRISPR activity. This method is easy to design and can be applied to various miRNAs via simple sequence manipulation. Therefore, this strategy provides a general platform for controlled genome editing.

RevDate: 2023-11-27
CmpDate: 2023-11-27

Jeung JH, Han H, Lee CY, et al (2023)

CRISPR/Cas12a Collateral Cleavage Activity for Sensitive 3'-5' Exonuclease Assay.

Biosensors, 13(11):.

This study presents a technique for detecting 3'-5' exonuclease activity through the use of CRISPR/Cas12a. These enzymes, including 3'-5' exonuclease (Exo III), perform crucial roles in various cellular processes and are associated with life expectancy. However, imbalances in their expression can increase susceptibility to diseases such as cancer, particularly under prolonged stress. In this study, an activator sequence of CRISPR/Cas12a was constructed on the 5'-end of a hairpin probe (HP), forming a blunt end. When the 3'-end of the HP was hydrolyzed with Exo III activity, the activator sequence of Cas12a was exposed, which led to collateral cleavage of the DNA signal probe and generated a fluorescent signal, allowing sensitive and highly specific Exo III detection. This detection principle relied on the fact that Exo III exclusively cleaves the 3'-end mononucleotide of dsDNA and does not affect ssDNA. Based on this strategy, Exo III activity was successfully assayed at 0.0073 U/mL, demonstrating high sensitivity. In addition, this technique was used to screen candidate inhibitors of Exo III activity.

RevDate: 2023-11-26

Joshi A, Yang SY, Song HG, et al (2023)

Genetic Databases and Gene Editing Tools for Enhancing Crop Resistance against Abiotic Stress.

Biology, 12(11):.

Abiotic stresses extensively reduce agricultural crop production globally. Traditional breeding technology has been the fundamental approach used to cope with abiotic stresses. The development of gene editing technology for modifying genes responsible for the stresses and the related genetic networks has established the foundation for sustainable agriculture against environmental stress. Integrated approaches based on functional genomics and transcriptomics are now expanding the opportunities to elucidate the molecular mechanisms underlying abiotic stress responses. This review summarizes some of the features and weblinks of plant genome databases related to abiotic stress genes utilized for improving crops. The gene-editing tool based on clustered, regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) has revolutionized stress tolerance research due to its simplicity, versatility, adaptability, flexibility, and broader applications. However, off-target and low cleavage efficiency hinder the successful application of CRISPR/Cas systems. Computational tools have been developed for designing highly competent gRNA with better cleavage efficiency. This powerful genome editing tool offers tremendous crop improvement opportunities, overcoming conventional breeding techniques' shortcomings. Furthermore, we also discuss the mechanistic insights of the CRISPR/Cas9-based genome editing technology. This review focused on the current advances in understanding plant species' abiotic stress response mechanism and applying the CRISPR/Cas system genome editing technology to develop crop resilience against drought, salinity, temperature, heavy metals, and herbicides.

RevDate: 2023-11-27
CmpDate: 2023-11-27

Cercenado E (2023)

What are the most relevant publications in Clinical Microbiology in the last two years?.

Revista espanola de quimioterapia : publicacion oficial de la Sociedad Espanola de Quimioterapia, 36 Suppl 1:64-67.

This minireview describes some of the articles published in the last two years related to innovative technologies including CRISPR-Cas, surface-enhanced Raman spectroscopy, microfluidics, flow cytometry, Fourier transform infrared spectroscopy, and artificial intelligence and their application to microbiological diagnosis, molecular typing and antimicrobial susceptibility testing. In addition, some articles related to resistance to new antimicrobials (ceftazidime-avibactam, meropenem-vaborbactam, imipenem-relebactam, and cefiderocol) are also described.

RevDate: 2023-11-27
CmpDate: 2023-11-27

Qiao Z, Xue L, Sun M, et al (2023)

Highly sensitive detection of Salmonella based on dual-functional HCR-mediated multivalent aptamer and amplification-free CRISPR/Cas12a system.

Analytica chimica acta, 1284:341998.

BACKGROUND: Salmonella infection severely threatens human health and causes substantial medical and financial concerns. Sensitive and specific detection of Salmonella in food samples is crucial but remains challenging. While some traditional assays for S. typhimurium are reliable, they suffer from various limitations, such as being time-consuming (culture-based methods), involving intricate nucleic molecular extraction (polymerization chain reaction, PCR), and exhibiting inadequate sensitivity (enzyme-linked immunosorbent assay, ELISA). In this case, it is essential to establish a rapid, simple-operation, and sensitive method for monitoring S. typhimurium to preserve food quality and prevent contamination.

RESULT: Herein, an amplification-free detection method for Salmonella was developed by coupling the aptamer magnetic separation with dual-functional HCR (hybridization chain reaction)-scaffold multivalent aptamer and the activity of CRISPR/Cas12a. In the detection system, the dual-functional HCR-scaffold multivalent aptamer with high binding affinity and specificity was fabricated in advance by assembling numerous Salmonella specific aptamers on the long HCR products. In addition to the enhanced affinity, the HCR-multiApt also contains a massive amount of repeated CRISPR-targetable DNA units in its HCR scaffold, which could trigger the trans-cleavage activity of Cas12a. In the presence of target bacteria, the HCR-scaffold multivalent aptamer could attach on the surface of bacteria effectively and amplified the signal of bacteria into CRISPR/Cas12a based fluorescent readout. The proposed detection system allowed for ultrasensitive detection of Salmonella in a linear range from 10[0] to 10[7] cfu mL[-1] with a LOD (limit of detection) of 2 cfu mL[-1].

SIGNIFICANCE: The novel dual-functional HCR-multiApt presents a simple and powerful strategy for improving the aptamer binding affinity toward Salmonella. Simultaneously, integrating this dual-functional HCR-multiApt with the CRISPR/Cas12a system significantly enhances the sensitivity by cascade signal amplification in a nucleic acids amplification-free way. Finally, leveraging the versatility of the aptamer, this highly sensitive method can be further extended for application in the detection of other bacteria, food safety monitoring, or clinical diagnostics.

RevDate: 2023-11-23

Tu W, Hu X, Wan R, et al (2023)

Effective delivery of miR-511-3p with mannose-decorated exosomes with RNA nanoparticles confers protection against asthma.

Journal of controlled release : official journal of the Controlled Release Society pii:S0168-3659(23)00751-4 [Epub ahead of print].

Our previous studies have shown that miR-511-3p treatment has a beneficial effect in alleviating allergic airway inflammation. Here, we sought to explore its therapeutic potential in animal models and gain a deeper understanding of its therapeutic value for asthma. miR-511-3p knockout mice (miR-511-3p[-/-]) were generated by CRISPR/Cas and showed exacerbated airway hyper-responsiveness and Th2-associated allergic airway inflammation compared with wild-type (WT) mice after exposed to cockroach allergen. RNA nanoparticles with mannose decorated EV-miR-511-3p were also created by loading miR-511-3p mimics into the mannose decorated EVs with engineered RNA nanoparticle PRNA-3WJ (Man-EV-miR-511-3p). Intra-tracheal inhalation of Man-EV-miR-511-3p, which could effectively penetrate the airway mucus barrier and deliver functional miR-511-3p to lung macrophages, successfully reversed the increased airway inflammation observed in miR-511-3p[-/-] mice. Through microarray analysis, complement C3 (C3) was identified as one of the major targets of miR-511-3p. C3 was increased in LPS-treated macrophages but decreased after miR-511-3p treatment. Consistent with these findings, C3 expression was elevated in the lung macrophages of an asthma mouse model but decreased in mice treated with miR-511-3p. Further experiments, including miRNA-mRNA pulldown and luciferase reporter assays, confirmed that miR-511-3p directly binds to C3 and activates the C3 gene. Thus, miR-511-3p represents a promising therapeutic target for asthma, and RNA nanotechnology reprogrammed EVs are efficient carriers for miRNA delivery for disease treatment.

RevDate: 2023-11-27
CmpDate: 2023-11-27

Altae-Tran H, Kannan S, Suberski AJ, et al (2023)

Uncovering the functional diversity of rare CRISPR-Cas systems with deep terascale clustering.

Science (New York, N.Y.), 382(6673):eadi1910.

Microbial systems underpin many biotechnologies, including CRISPR, but the exponential growth of sequence databases makes it difficult to find previously unidentified systems. In this work, we develop the fast locality-sensitive hashing-based clustering (FLSHclust) algorithm, which performs deep clustering on massive datasets in linearithmic time. We incorporated FLSHclust into a CRISPR discovery pipeline and identified 188 previously unreported CRISPR-linked gene modules, revealing many additional biochemical functions coupled to adaptive immunity. We experimentally characterized three HNH nuclease-containing CRISPR systems, including the first type IV system with a specified interference mechanism, and engineered them for genome editing. We also identified and characterized a candidate type VII system, which we show acts on RNA. This work opens new avenues for harnessing CRISPR and for the broader exploration of the vast functional diversity of microbial proteins.

RevDate: 2023-11-23

Jibrilla M, Raji H, MI Okeke (2023)

Survey of attitude to human genome modification in Nigeria.

Journal of community genetics [Epub ahead of print].

Gene editing and mitochondrial replacement therapy (MRT) are biotechnologies used to modify the host nuclear and mitochondrial DNA, respectively. Gene editing is the modification of a region of the host genome using site-specific nucleases, in particular the clustered regularly interspaced short palindromic repeats (CRISPR)-Cas system. Heritable and somatic genome editing (HGE and SGE) are used in gene therapy. MRT is a technique used to substitute the defective mitochondria in the recipient embryo with a female donor healthy mitochondrion in order to prevent the inheritance of mothers' defective mitochondria resulting in the change of mitochondria of the entire generation to come. To evaluate the perception of the Nigerian citizens on human genome modification, two survey forms were created and distributed in-person and majorly online. There was a total of 268 responses, 188 from the public and 80 from health workers and bio-scientists. The results showed poor knowledge about gene editing and MRT by the Nigerian public, but its use to prevent and cure inherited diseases was supported. Morality and religion have great influence on the attitude of Nigerians towards genome modification, but the influence of religion and morality is not unequivocal. Multiple regression analysis of Nigerian public responses shows that gender (females), age (19-30 years), monthly income (NGN 0 to 30,000), and level of education (tertiary) are significantly associated with approval of human genome editing, but the survey of health workers and bio-scientists shows no significant association except for females who approve and Muslims who disapprove of human genome editing.

RevDate: 2023-11-25
CmpDate: 2023-11-24

Hu Y, Liu L, Jiang Q, et al (2023)

CRISPR/Cas9: a powerful tool in colorectal cancer research.

Journal of experimental & clinical cancer research : CR, 42(1):308.

Colorectal cancer (CRC) is one of the most common malignant cancers worldwide and seriously threatens human health. The clustered regulatory interspaced short palindromic repeat/CRISPR-associate nuclease 9 (CRISPR/Cas9) system is an adaptive immune system of bacteria or archaea. Since its introduction, research into various aspects of treatment approaches for CRC has been accelerated, including investigation of the oncogenes, tumor suppressor genes (TSGs), drug resistance genes, target genes, mouse model construction, and especially in genome-wide library screening. Furthermore, the CRISPR/Cas9 system can be utilized for gene therapy for CRC, specifically involving in the molecular targeted drug delivery or targeted knockout in vivo. In this review, we elucidate the mechanism of the CRISPR/Cas9 system and its comprehensive applications in CRC. Additionally, we discussed the issue of off-target effects associated with CRISPR/Cas9, which serves to restrict its practical application. Future research on CRC should in-depth and systematically utilize the CRISPR/Cas9 system thereby achieving clinical practice.

RevDate: 2023-11-25
CmpDate: 2023-11-24

Kouroukli AG, Rajaram N, Bashtrykov P, et al (2023)

Targeting oncogenic TERT promoter variants by allele-specific epigenome editing.

Clinical epigenetics, 15(1):183.

BACKGROUND: Activation of dominant oncogenes by small or structural genomic alterations is a common driver mechanism in many cancers. Silencing of such dominantly activated oncogenic alleles, thus, is a promising strategy to treat cancer. Recently, allele-specific epigenome editing (ASEE) has been described as a means to reduce transcription of genes in an allele-specific manner. In cancer, specificity to an oncogenic allele can be reached by either targeting directly a pathogenic single-nucleotide variant or a polymorphic single-nucleotide variant linked to the oncogenic allele. To investigate the potential of ASEE in cancer, we here explored this approach by targeting variants at the TERT promoter region. The TERT promoter region has been described as one of the most frequently mutated non-coding cancer drivers.

RESULTS: Sequencing of the TERT promoter in cancer cell lines showed 53% (41/77) to contain at least one heterozygous sequence variant allowing allele distinction. We chose the hepatoblastoma cell line Hep-G2 and the lung cancer cell line A-549 for this proof-of-principle study, as they contained two different kinds of variants, namely the activating mutation C228T in the TERT core promoter and the common SNP rs2853669 in the THOR region, respectively. These variants were targeted in an allele-specific manner using sgRNA-guided dCas9-DNMT3A-3L complexes. In both cell lines, we successfully introduced DNA methylation specifically to the on-target allele of the TERT promoter with limited background methylation on the off-target allele or an off-target locus (VEGFA), respectively. We observed a maximum CpG methylation gain of 39% and 76% on the target allele when targeting the activating mutation and the common SNP, respectively. The epigenome editing translated into reduced TERT RNA expression in Hep-G2.

CONCLUSIONS: We applied an ASEE-mediated approach to silence TERT allele specifically. Our results show that the concept of dominant oncogene inactivation by allele-specific epigenome editing can be successfully translated into cancer models. This new strategy may have important advantages in comparison with existing therapeutic approaches, e.g., targeting telomerase, especially with regard to reducing adverse side effects.

RevDate: 2023-11-27
CmpDate: 2023-11-27

Song Y (2023)

RNA-based anti-CRISPRs.

Nature chemical biology, 19(12):1433.

RevDate: 2023-11-22

Zaman QU, Raza A, Lozano-Juste J, et al (2023)

Engineering plants using diverse CRISPR-associated proteins and deregulation of genome-edited crops.

Trends in biotechnology pii:S0167-7799(23)00298-6 [Epub ahead of print].

The CRISPR/Cas system comprises RNA-guided nucleases, the target specificity of which is directed by Watson-Crick base pairing of target loci with single guide (sg)RNA to induce the desired edits. CRISPR-associated proteins and other engineered nucleases are opening new avenues of research in crops to induce heritable mutations. Here, we review the diversity of CRISPR-associated proteins and strategies to deregulate genome-edited (GEd) crops by considering them to be close to natural processes. This technology ensures yield without penalties, advances plant breeding, and guarantees manipulation of the genome for desirable traits. DNA-free and off-target-free GEd crops with defined characteristics can help to achieve sustainable global food security under a changing climate, but need alignment of international regulations to operate in existing supply chains.

RevDate: 2023-11-25

Wen J, Deng H, He D, et al (2023)

Dual-functional DNAzyme powered CRISPR-Cas12a sensor for ultrasensitive and high-throughput detection of Pb[2+] in freshwater.

The Science of the total environment, 911:168708 pii:S0048-9697(23)07336-9 [Epub ahead of print].

Freshwater lead pollution has posed severe threat to the environment and human health, underscoring the urgent necessity for accurate and user-friendly detection methods. Herein, we introduce a novel Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR-Cas) sensor for highly sensitive Pb[2+] detection. To accomplish this, we designed a dual-functional deoxyribozyme (df-DNAzyme) probe that functions as an activator for the CRISPR-Cas12a system while also recognizing Pb[2+]. The df-DNAzyme probe was subsequently combined with gold nanoparticles (AuNPs) to fabricate a DNAzyme/AuNP nanoprobe, facilitating the activation of CRISPR-Cas12a in a one-to-multiple manner. Upon exposure to Pb[2+], the df-DNAzyme is cleaved, causing disintegration of the DNAzyme/AuNP nanoprobe from magnetic beads. The degraded DNAzyme/AuNP containing multiple double-stranded DNA activators efficiently triggers CRISPR-Cas12a activity, initiating cleavage of fluorescence-quenched reporter DNA and generating amplified signals accordingly. The amplified fluorescence signal is accurately quantified using a quantitative polymerase chain reaction (qPCR) instrument capable of measuring 96 or 384 samples simultaneously at the microliter scale. This technique demonstrates ultra-sensitive detection capability for Pb[2+] at concentrations as low as 1 pg/L within a range from 1 pg/L to 10 μg/L, surpassing limits set by World Health Organization (WHO) and United States Environmental Protection Agency (US EPA) guidelines. This study offers an ultrasensitive and high-throughput method for the detection of Pb[2+] in freshwater, thereby advancing a novel approach towards the development of precise and convenient techniques for detecting harmful contaminants.

RevDate: 2023-11-22

Yirmiya E, Leavitt A, Lu A, et al (2023)

Phages overcome bacterial immunity via diverse anti-defence proteins.

Nature pii:10.1038/s41586-023-06869-w [Epub ahead of print].

It was recently shown that bacteria employ, apart from CRISPR-Cas and restriction systems, a considerable diversity of phage resistance systems[1-4], but it is largely unknown how phages cope with this multilayered bacterial immunity. Here, we analyzed groups of closely related Bacillus phages that showed differential sensitivity to bacterial defense systems, and discovered four distinct families of anti-defense proteins that inhibit the Gabija, Thoeris, and Hachiman systems. We show that these proteins Gad1, Gad2, Tad2, and Had1 efficiently cancel the defensive activity when co-expressed with the respective defense system or introduced into phage genomes. Homologs of these anti-defense proteins are found in hundreds of phages that infect taxonomically diverse bacterial species. We show that the anti-Gabija protein Gad1 blocks the ability of the Gabija defense complex to cleave phage-derived DNA. Our data further reveal an anti-Thoeris protein, denoted Tad2, which is a "sponge" that sequesters the immune signaling molecules produced by Thoeris TIR-domain proteins in response to phage. Our results demonstrate that phages encode an arsenal of anti-defense proteins that can disable a variety of bacterial defense mechanisms.

RevDate: 2023-11-27
CmpDate: 2023-11-27

Limaye A, Cho K, Hall B, et al (2023)

Genotyping Protocols for Genetically Engineered Mice.

Current protocols, 3(11):e929.

Historically, the laboratory mouse has been the mammalian species of choice for studying gene function and for modeling diseases in humans. This was mainly due to their availability from mouse fanciers. In addition, their short generation time, small size, and minimal food consumption compared to that of larger mammals were definite advantages. This led to the establishment of large hubs for the development of genetically modified mouse models, such as the Jackson Laboratory. Initial research into inbred mouse strains in the early 1900s revolved around coat color genetics and cancer studies, but gene targeting in embryonic stem cells and the introduction of transgenes through pronuclear injection of a mouse zygote, along with current clustered regularly interspaced short palindromic repeat (CRISPR) RNA gene editing, have allowed easy manipulation of the mouse genome. Originally, to distribute a mouse model to other facilities, standard methods had to be developed to ensure that each modified mouse trait could be consistently identified no matter which laboratory requested it. The task of establishing uniform protocols became easier with the development of the polymerase chain reaction (PCR). This chapter will provide guidelines for identifying genetically modified mouse models, mainly using endpoint PCR. In addition, we will discuss strategies to identify genetically modified mouse models that have been established using newer gene-editing technology such as CRISPR. Published 2023. This article is a U.S. Government work and is in the public domain in the USA. Basic Protocol 1: Digestion with proteinase K followed by purification of genomic DNA using phenol/chloroform Alternate Protocol: Digestion with proteinase K followed by crude isopropanol extraction of genomic DNA for tail biopsy and ear punch samples Basic Protocol 2: Purification of genomic DNA using a semi-automated system Basic Protocol 3: Purification of genomic DNA from semen, blood, or buccal swabs Basic Protocol 4: Purification of genomic DNA from mouse blastocysts to assess CRISPR gene editing Basic Protocol 5: Routine endpoint-PCR-based genotyping using DNA polymerase and thermal cycler Basic Protocol 6: T7E1/Surveyor assays to detect insertion or deletions following CRISPR editing Basic Protocol 7: Detecting off-target mutations following CRISPR editing Basic Protocol 8: Detecting genomic sequence deletion after CRISPR editing using a pair of guide RNAs Basic Protocol 9: Detecting gene knock-in events following CRISPR editing Basic Protocol 10: Screening of conditional knockout floxed mice.

RevDate: 2023-11-27
CmpDate: 2023-11-27

Rueff AS, van Raaphorst R, Aggarwal SD, et al (2023)

Synthetic genetic oscillators demonstrate the functional importance of phenotypic variation in pneumococcal-host interactions.

Nature communications, 14(1):7454.

Phenotypic variation is the phenomenon in which clonal cells display different traits even under identical environmental conditions. This plasticity is thought to be important for processes including bacterial virulence, but direct evidence for its relevance is often lacking. For instance, variation in capsule production in the human pathogen Streptococcus pneumoniae has been linked to different clinical outcomes, but the exact relationship between variation and pathogenesis is not well understood due to complex natural regulation. In this study, we use synthetic oscillatory gene regulatory networks (GRNs) based on CRISPR interference (CRISPRi) together with live cell imaging and cell tracking within microfluidics devices to mimic and test the biological function of bacterial phenotypic variation. We provide a universally applicable approach for engineering intricate GRNs using only two components: dCas9 and extended sgRNAs (ext-sgRNAs). Our findings demonstrate that variation in capsule production is beneficial for pneumococcal fitness in traits associated with pathogenesis providing conclusive evidence for this longstanding question.

RevDate: 2023-11-27
CmpDate: 2023-11-27

Liang P, Lv B, Chen K, et al (2023)

An ultrasensitive Cd[2+] detection biosensor based on DNAzyme and CRISPR/Cas12a coupled with hybridization chain reaction.

Analytica chimica acta, 1283:341950.

The detection of cadmium is essential because it poses a significant threat to human health and the environment. Recent advancements in biosensors that detect nonnucleic-acid targets using CRISPR/Cas12a in combination with aptamers or DNAzymes show promising performance. Herein, we integrated DNAzyme, hybridization chain reaction (HCR) and CRISPR/Cas12a into a single biosensor for the first time and realized the ultrasensitive detection of Cd[2+]. A single phosphorothioate ribonucleobase (rA)-containing oligonucleotide (PS substrate) and a Cd[2+]-specific DNAzyme (Cdzyme) are used for Cd[2][+] recognition, releasing short single-stranded DNA. Then, the HCR is triggered by the cleavage products for signal transduction and amplification. Next, the trans-cleavage activity of Cas12a is activated due to the presence of crRNA complementary strands and PAM sites in the HCR products. As a result, FQ-reporters are cleaved, and the fluorescence values can be easily read using a fluorometer, allowing Cd[2][[+]] quantification by measuring the fluorescent signal. The Cd[2][[+]] detection biosensor is ultrasensitive with a detection limit of 1.25 pM. Moreover, the biosensor shows great stability under different pH and various anion conditions. The proposed sensor was utilized for environmental water sample detection, demonstrating the dependability of the detection system. Considering the high sensitivity and reliable performance of the assay, it could be further used in environmental monitoring. In addition, the design strategy reported in this study could extend the application of CRISPR/Cas12a in heavy metal detection.

RevDate: 2023-11-27
CmpDate: 2023-11-27

Benarroch L (2023)

[CRIPSR-Cas9: A therapeutic strategy for laminopathies?].

Medecine sciences : M/S, 39 Hors série n° 1:65.

RevDate: 2023-11-27
CmpDate: 2023-11-27

Naddaf M (2023)

First trial of 'base editing' in humans lowers cholesterol - but raises safety concerns.

Nature, 623(7988):671-672.

RevDate: 2023-11-27
CmpDate: 2023-11-27

Yudin Kharismasari C, Irkham , Zein MIHL, et al (2024)

CRISPR/Cas12-based electrochemical biosensors for clinical diagnostic and food monitoring.

Bioelectrochemistry (Amsterdam, Netherlands), 155:108600.

Each organism has a unique sequence of nitrogenous bases in in the form of DNA or RNA which distinguish them from other organisms. This characteristic makes nucleic acid-based detection extremely selective and compare to other molecular techniques. In recent years, several nucleic acid-based detection technology methods have been developed, one of which is the electrochemical biosensor. Electrochemical biosensors are known to have high sensitivity and accuracy. In addition, the ease of miniaturization of this electrochemical technique has garnered interest from many researchers. On the other hand, the CRISPR/Cas12 method has been widely used in detecting nucleic acids due to its highly selective nature. The CRISPR/Cas12 method is also reported to increase the sensitivity of electrochemical biosensors through the utilization of modified electrodes. The electrodes can be modified according to detection needs so that the biosensor's performance can be improved. This review discusses the application of CRISPR/Cas12-based electrochemical biosensors, as well as various electrode modifications that have been successfully used to improve the performance of these biosensors in the clinical and food monitoring fields.

RevDate: 2023-11-27
CmpDate: 2023-11-27

Shaw WM, Khalil AS, T Ellis (2023)

A Multiplex MoClo Toolkit for Extensive and Flexible Engineering of Saccharomyces cerevisiae.

ACS synthetic biology, 12(11):3393-3405.

Synthetic biology toolkits are one of the core foundations on which the field has been built, facilitating and accelerating efforts to reprogram cells and organisms for diverse biotechnological applications. The yeast Saccharomyces cerevisiae, an important model and industrial organism, has benefited from a wide range of toolkits. In particular, the MoClo Yeast Toolkit (YTK) enables the fast and straightforward construction of multigene plasmids from a library of highly characterized parts for programming new cellular behavior in a more predictable manner. While YTK has cultivated a strong parts ecosystem and excels in plasmid construction, it is limited in the extent and flexibility with which it can create new strains of yeast. Here, we describe a new and improved toolkit, the Multiplex Yeast Toolkit (MYT), that extends the capabilities of YTK and addresses strain engineering limitations. MYT provides a set of new integration vectors and selectable markers usable across common laboratory strains, as well as additional assembly cassettes to increase the number of transcriptional units in multigene constructs, CRISPR-Cas9 tools for highly efficient multiplexed vector integration, and three orthogonal and inducible promoter systems for conditional programming of gene expression. With these tools, we provide yeast synthetic biologists with a powerful platform to take their engineering ambitions to exciting new levels.

RevDate: 2023-11-27
CmpDate: 2023-11-27

Chen X, Huang C, Zhang J, et al (2024)

Mini crRNA-mediated CRISPR/Cas12a system (MCM-CRISPR/Cas12a) and its application in RNA detection.

Talanta, 268(Pt 1):125350.

Some non-coding RNAs are abnormally expressed during the occurrence and development of diseases, so it is necessary to develop analytical methods that can specifically and sensitively detect them. In typical CRISPR/Cas12a system, a complete crRNA that can recognize single-stranded or double-stranded DNA is necessary to activate its trans-cleavage activity, which limits its direct application in RNA detection. Here, we prospectively find that slicing the facilitated crRNA in the typical CRISPR/Cas12a system at a fitted site did not affect its trans-cleavage activity, and a mini crRNA-mediated CRISPR/Cas12a system (MCM-CRISPR/Cas12a) was proposed based on this. This system can detect non-coding RNA to pM-level (10 pM for miRNA-21). To expand the application of this system, we combined it with HCR and CHA to establish a detection platform for non-coding RNA. The results show that the proposed method can specifically detect RNA to fM-level (2.5 fM for miRNA-21, 8.98 fM for miR-128-3p, and 81.6 fM for lncRNA PACER). The spiked recovery rates of miRNA-21, miR-128-3p, and lncRNA PACER in normal human serum were in range from 104.7 to 109.4 %, indicating the proposed method owns good applicability. In general, this MCM-CRISPR/Cas12a system further breaks the limitations of the typical CRISPR/Cas12a system that cannot be directly used for non-coding RNA detection. Besides, its combination with HCR and CHA achieves highly sensitive detection of non-coding RNA.

RevDate: 2023-11-27
CmpDate: 2023-11-27

Feng Q, Hao S, Fang P, et al (2023)

Role of GPX4 inhibition-mediated ferroptosis in the chemoresistance of ovarian cancer to Taxol in vitro.

Molecular biology reports, 50(12):10189-10198.

BACKGROUND: Ovarian cancer remains a common gynecological tumor and the fifth leading cause of death worldwide. Taxol-based chemotherapy is a standard approach to the treatment of ovarian cancer. Glutathione peroxidase 4 (GPX4) is the key regulator of ferroptosis, which is an important form of cell death. Here, we investigate the effect of GPX4 inhibition-mediated ferroptosis on the sensitivity of ovarian cancer cells to Taxol.

METHODS AND RESULTS: A2780/PTX and OVCAR-3/PTX Taxol-resistant ovarian cancer cells were established, and stable GPX4 knockout cell lines were generated via lentivirus GPX4-sgRNA. The GPX4 expression level, the apoptosis rate and cell viability were analyzed. The levels of ferroptosis-related factor indicators such as malondialdehyde (MDA) and reactive oxygen species (ROS) were measured. The results showed that the GPX4 protein and mRNA levels were increased in the Taxol-resistant cells. Moreover, GPX4 knockout reduced cell viability and inhibited the colony formation rate. In addition, we found that GPX4 inhibition increased Taxol sensitivity by inducing ferroptosis.

CONCLUSIONS: In summary, our studies reveal that GPX4 inhibition promotes ferroptosis and increases the sensitivity of ovarian cancer cells to Taxol in vitro.

RevDate: 2023-11-27
CmpDate: 2023-11-27

Li T, N Cheng (2023)

Sensitive and Portable Signal Readout Strategies Boost Point-of-Care CRISPR/Cas12a Biosensors.

ACS sensors, 8(11):3988-4007.

Point-of-care (POC) detection is getting more and more attention in many fields due to its accuracy and on-site test property. The CRISPR/Cas12a system is endowed with excellent sensitivity, target identification specificity, and signal amplification ability in biosensing because of its unique trans-cleavage ability. As a result, a lot of research has been made to develop CRISPR/Cas12a-based biosensors. In this review, we focused on signal readout strategies and summarized recent sensitivity-improving strategies in fluorescence, colorimetric, and electrochemical signaling. Then we introduced novel portability-improving strategies based on lateral flow assays (LFAs), microfluidic chips, simplified instruments, and one-pot design. In the end, we also provide our outlook for the future development of CRISPR/Cas12a biosensors.


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.

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By delivering the Cas9 nuclease, complexed with a synthetic guide RNA (gRNA) into a cell, the cell's genome can be precisely cut at any desired location, allowing existing genes to be removed and/or new ones added. That is, the CRISPR-Cas system provides a tool for the cut-and-paste editing of genomes. Welcome to the brave new world of genome editing. R. Robbins

Electronic Scholarly Publishing
961 Red Tail Lane
Bellingham, WA 98226

E-mail: RJR8222 @

Papers in Classical Genetics

The ESP began as an effort to share a handful of key papers from the early days of classical genetics. Now the collection has grown to include hundreds of papers, in full-text format.

Digital Books

Along with papers on classical genetics, ESP offers a collection of full-text digital books, including many works by Darwin (and even a collection of poetry — Chicago Poems by Carl Sandburg).


ESP now offers a much improved and expanded collection of timelines, designed to give the user choice over subject matter and dates.


Biographical information about many key scientists.

Selected Bibliographies

Bibliographies on several topics of potential interest to the ESP community are now being automatically maintained and generated on the ESP site.

ESP Picks from Around the Web (updated 07 JUL 2018 )