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Bibliography on: CRISPR-Cas

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ESP: PubMed Auto Bibliography 25 Oct 2020 at 01:39 Created: 

CRISPR-Cas

Clustered regularly interspaced short palindromic repeats (CRISPR, pronounced crisper) are segments of prokaryotic DNA containing short repetitions of base sequences. Each repetition is followed by short segments of "spacer DNA" from previous exposures to foreign DNA (e.g a virus or plasmid). The CRISPR/Cas system is a prokaryotic immune system that confers resistance to foreign genetic elements such as those present within plasmids and phages, and provides a form of acquired immunity. CRISPR associated proteins (Cas) use the CRISPR spacers to recognize and cut these exogenous genetic elements in a manner analogous to RNA interference in eukaryotic organisms. CRISPRs are found in approximately 40% of sequenced bacterial genomes and 90% of sequenced archaea. By delivering the Cas9 nuclease complexed with a synthetic guide RNA (gRNA) into a cell, the cell's genome can be cut at a desired location, allowing existing genes to be removed and/or new ones added. The Cas9-gRNA complex corresponds with the CAS III crRNA complex in the above diagram. CRISPR/Cas genome editing techniques have many potential applications, including altering the germline of humans, animals, and food crops. The use of CRISPR Cas9-gRNA complex for genome editing was the AAAS's choice for breakthrough of the year in 2015.

Created with PubMed® Query: "CRISPR.CAS" OR "crispr/cas" NOT pmcbook NOT ispreviousversion

Citations The Papers (from PubMed®)

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RevDate: 2020-10-24

Matsumoto D, Tamamura H, W Nomura (2020)

A cell cycle-dependent CRISPR-Cas9 activation system based on an anti-CRISPR protein shows improved genome editing accuracy.

Communications biology, 3(1):601 pii:10.1038/s42003-020-01340-2.

The development of genome editing systems based on the Cas9 endonuclease has greatly facilitated gene knockouts and targeted genetic alterations. Precise editing of target genes without off-target effects is crucial to prevent adverse effects in clinical applications. Although several methods have been reported to result in less off-target effects associated with the CRISPR technology, these often exhibit lower editing efficiency. Therefore, efficient, accurate, and innocuous CRISPR technology is still required. Anti-CRISPR proteins are natural inhibitors of CRISPR-Cas systems derived from bacteriophages. Here, the anti-CRISPR protein, AcrIIA4, was fused with the N terminal region of human Cdt1 that is degraded specifically in S and G2, the phases of the cell cycle when homology-directed repair (HDR) is dominant. Co-expression of SpyCas9 and AcrIIA4-Cdt1 not only increases the frequency of HDR but also suppress off-targets effects. Thus, the combination of SpyCas9 and AcrIIA4-Cdt1 is a cell cycle-dependent Cas9 activation system for accurate and efficient genome editing.

RevDate: 2020-10-24

García-Moyano A, Larsen Ø, Gaykawad S, et al (2020)

Fragment exchange (FX) plasmid tools for CRISPR/Cas9-mediated gene integration and protease production in Bacillus subtilis.

Applied and environmental microbiology pii:AEM.02090-20 [Epub ahead of print].

Since its discovery as part of the bacterial adaptative immune system, CRISPR/Cas has emerged as the most promising tool on targeted genome-editing over the past few years. Various tools for genome editing in Bacillus subtilis have recently been developed, expanding and simplifying its potential development as an industrial strain. A collection of vectors compatible with high-throughput fragment exchange (FX) cloning for heterologous expression in E. coli and Bacillus were previously developed. This vector catalogue was through this work supplemented with editing plasmid for genome engineering in Bacillus by adapting two CRISPR/Cas plasmids to the cloning technology. The customized tools allow versatile editing at any chosen genomic position (single-plasmid strategy) or at a fixed genomic locus (double-plasmid strategy). The single-plasmid strategy was validated by deleting the spoIIAC gene, which has an essential role in sporulation. Using the double-plasmid strategy, we demonstrate the quick transition from plasmid-based subtilisin expression to a stable integration of the gene into the amyE locus of a seven-protease deficient KO7 strain. The newly engineered B. subtilis strain allowed for a successful production of a functional enzyme. The customized tools provide improvements to the cloning procedure, should be useful for versatile genomic engineering and contributes to a cloning platform for quick transition from HTP enzyme expression to production through fermentation of the industrially relevant B. subtilis and related strains.Importance We complemented a cloning platform with new editing plasmids that allows a quick transition from high-throughput cloning and expression of new enzymes to stable integration of genes for production of enzymes through B. subtilis fermentation. We present two systems for the effective assembly cloning of any genome editing cassette that shortens the engineering procedure to obtain the final editing constructs. The utility of the customized tools is demonstrated by disrupting Bacillus' capacity to sporulate, and by introducing stable expression of subtilisin. The tools should be useful to engineered B. subtilis strains by a variety of recombination events to ultimately improve the application range of this industry relevant host.

RevDate: 2020-10-23

Bertelsen MB, Senissar M, Nielsen MH, et al (2020)

Structural Basis for Toxin Inhibition in the VapXD Toxin-Antitoxin System.

Structure (London, England : 1993) pii:S0969-2126(20)30373-7 [Epub ahead of print].

Bacterial type II toxin-antitoxin (TA) modules encode a toxic protein that downregulates metabolism and a specific antitoxin that binds and inhibits the toxin during normal growth. In non-typeable Haemophilus influenzae, a common cause of infections in humans, the vapXD locus was found to constitute a functional TA module and contribute to pathogenicity; however, the mode of action of VapD and the mechanism of inhibition by the VapX antitoxin remain unknown. Here, we report the structure of the intact H. influenzae VapXD complex, revealing an unusual 2:1 TA molecular stoichiometry where a Cas2-like homodimer of VapD binds a single VapX antitoxin. VapX consists of an oligonucleotide/oligosaccharide-binding domain that docks into an asymmetrical cavity on the toxin dimer. Structures of isolated VapD further reveal how a symmetrical toxin homodimer adapts to interacting with an asymmetrical antitoxin and suggest how a primordial TA system evolved to become part of CRISPR-Cas immunity systems.

RevDate: 2020-10-23

Galizi R, Duncan JN, Rostain W, et al (2020)

Engineered RNA-Interacting CRISPR Guide RNAs for Genetic Sensing and Diagnostics.

The CRISPR journal, 3(5):398-408.

CRISPR guide RNAs (gRNAs) can be programmed with relative ease to allow the genetic editing of nearly any DNA or RNA sequence. Here, we propose novel molecular architectures to achieve RNA-dependent modulation of CRISPR activity in response to specific RNA molecules. We designed and tested, in both living Escherichia coli cells and cell-free assays for rapid prototyping, cis-repressed RNA-interacting guide RNA (igRNA) that switch to their active state only upon interaction with small RNA fragments or long RNA transcripts, including pathogen-derived mRNAs of medical relevance such as the human immunodeficiency virus infectivity factor. The proposed CRISPR-igRNAs are fully customizable and easily adaptable to the majority if not all the available CRISPR-Cas variants to modulate a variety of genetic functions in response to specific cellular conditions, providing orthogonal activation and increased specificity. We thereby foresee a large scope of application for therapeutic, diagnostic, and biotech applications in both prokaryotic and eukaryotic systems.

RevDate: 2020-10-23

Wiegand T, Semenova E, Shiriaeva A, et al (2020)

Reproducible Antigen Recognition by the Type I-F CRISPR-Cas System.

The CRISPR journal, 3(5):378-387.

CRISPR-associated proteins 1 and 2 (Cas1-2) are necessary and sufficient for new spacer acquisition in some CRISPR-Cas systems (e.g., type I-E), but adaptation in other systems (e.g., type II-A) involves the crRNA-guided surveillance complex. Here we show that the type I-F Cas1-2/3 proteins are necessary and sufficient to produce low levels of spacer acquisition, but the presence of the type I-F crRNA-guided surveillance complex (Csy) improves the efficiency of adaptation and significantly increases the fidelity of protospacer adjacent motif selection. Sequences selected for integration are preferentially derived from specific regions of extrachromosomal DNA, and patterns of spacer selection are highly reproducible between independent biological replicates. This work helps define the role of the Csy complex in I-F adaptation and reveals that actively replicating mobile genetic elements have antigenic signatures that facilitate their integration during CRISPR adaptation.

RevDate: 2020-10-23

Polkoff KM, Chung J, Simpson SG, et al (2020)

In Vitro Validation of Transgene Expression in Gene-Edited Pigs Using CRISPR Transcriptional Activators.

The CRISPR journal, 3(5):409-418.

The use of CRISPR-Cas and RNA-guided endonucleases has drastically changed research strategies for understanding and exploiting gene function, particularly for the generation of gene-edited animal models. This has resulted in an explosion in the number of gene-edited species, including highly biomedically relevant pig models. However, even with error-free DNA insertion or deletion, edited genes are occasionally not expressed and/or translated as expected. Therefore, there is a need to validate the expression outcomes gene modifications in vitro before investing in the costly generation of a gene-edited animal. Unfortunately, many gene targets are tissue specific and/or not expressed in cultured primary cells, making validation difficult without generating an animal. In this study, using pigs as a proof of concept, we show that CRISPR-dCas9 transcriptional activators can be used to validate functional transgene insertion in nonexpressing easily cultured cells such as fibroblasts. This is a tool that can be used across disciplines and animal species to save time and resources by verifying expected outcomes of gene edits before generating live animals.

RevDate: 2020-10-23

Casas-Mollano JA, Zinselmeier MH, Erickson SE, et al (2020)

CRISPR-Cas Activators for Engineering Gene Expression in Higher Eukaryotes.

The CRISPR journal, 3(5):350-364.

CRISPR-Cas-based transcriptional activators allow genetic engineers to specifically induce expression of one or many target genes in trans. Here we review the many design variations of these versatile tools and compare their effectiveness in different eukaryotic systems. Lastly, we highlight several applications of programmable transcriptional activation to interrogate and engineer complex biological processes.

RevDate: 2020-10-23

Sudheer S, Bai RG, Usmani Z, et al (2020)

Insights on Engineered Microbes in Sustainable Agriculture: Biotechnological Developments and Future Prospects.

Current genomics, 21(5):321-333.

Background: Enhanced agricultural production is essential for increasing demand of the growing world population. At the same time, to combat the adverse effects caused by conventional agriculture practices to the environment along with the impact on human health and food security, a sustainable and healthy agricultural production needs to be practiced using beneficial microorganisms for enhanced yield. It is quite challenging because these microorganisms have rich biosynthetic repositories to produce biomolecules of interest; however, the intensive research in allied sectors and emerging genetic tools for improved microbial consortia are accepting new approaches that are helpful to farmers and agriculturists to meet the ever-increasing demand of sustainable food production. An important advancement is improved strain development via genetically engineered microbial systems (GEMS) as well as genetically modified microorganisms (GMOs) possessing known and upgraded functional characteristics to promote sustainable agriculture and food security. With the development of novel technologies such as DNA automated synthesis, sequencing and influential computational tools, molecular biology has entered the systems biology and synthetic biology era. More recently, CRISPR/Cas has been engineered to be an important tool in genetic engineering for various applications in the agri sector. The research in sustainable agriculture is progressing tremendously through GMOs/GEMS for their potential use in biofertilizers and as biopesticides.

Conclusion: In this review, we discuss the beneficial effects of engineered microorganisms through integrated sustainable agriculture production practices to improve the soil microbial health in order to increase crop productivity.

RevDate: 2020-10-23
CmpDate: 2020-10-23

Tyagi S, Kumar R, Kumar V, et al (2021)

Engineering disease resistant plants through CRISPR-Cas9 technology.

GM crops & food, 12(1):125-144.

Plants are susceptible to phytopathogens, including bacteria, fungi, and viruses, which cause colossal financial shortfalls (pre- and post-harvest) and threaten global food safety. To combat with these phytopathogens, plant possesses two-layer of defense in the form of PAMP-triggered immunity (PTI), or Effectors-triggered immunity (ETI). The understanding of plant-molecular interactions and revolution of high-throughput molecular techniques have opened the door for innovations in developing pathogen-resistant plants. In this context, Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) has transformed genome editing (GE) technology and being harnessed for altering the traits. Here we have summarized the complexities of plant immune system and the use of CRISPR-Cas9 to edit the various components of plant immune system to acquire long-lasting resistance in plants against phytopathogens. This review also sheds the light on the limitations of CRISPR-Cas9 system, regulation of CRISPR-Cas9 edited crops and future prospective of this technology.

RevDate: 2020-10-23
CmpDate: 2020-10-23

Hameed A, Hussain SA, Ijaz MU, et al (2020)

Deletions of the Idh1, Eco1, Rom2, and Taf10 Genes Differently Control the Hyphal Growth, Drug Tolerance, and Virulence of Candida albicans.

Folia biologica, 66(3):91-103.

The most recent genome-editing system called CRISPR-Cas9 (clustered regularly interspaced short palindromic repeat system with associated protein 9-nuclease) was employed to delete four non-essential genes (i.e., Caeco1, Caidh1, Carom2, and Cataf10) individually to establish their gene functionality annotations in pathogen Candida albicans. The biological roles of these genes were investigated with respect to the cell wall integrity and biogenesis, calcium/calcineurin pathways, susceptibility of mutants towards temperature, drugs and salts. All the mutants showed increased vulnerability compared to the wild-type background strain towards the cell wall-perturbing agents, (antifungal) drugs and salts. All the mutants also exhibited repressed and defective hyphal growth and smaller colony size than control CA14. The cell cycle of all the mutants decreased enormously except for those with Carom2 deletion. The budding index and budding size also increased for all mutants with altered bud shape. The disposition of the mutants towards cell wall-perturbing enzymes disclosed lower survival and more rapid cell wall lysis events than in wild types. The pathogenicity and virulence of the mutants was checked by adhesion assay, and strains lacking rom2 and eco1 were found to possess the least adhesion capacity, which is synonymous to their decreased pathogenicity and virulence.

RevDate: 2020-10-23
CmpDate: 2020-10-23

Chan JD, Beavis PA, PK Darcy (2020)

p38 Kinase: A Key Target for Driving Potent T Cells for Adoptive Immunotherapy.

Cancer cell, 37(6):756-758.

In this issue of Cancer Cell, Gurusamy et al. use a CRISPR-Cas9 screening approach to demonstrate that deletion of p38 increases multiple phenotypic qualities of effective anti-tumor T cells. Preconditioning T cells with a p38 inhibitor enhances anti-tumor efficacy of adoptive immunotherapy.

RevDate: 2020-10-23
CmpDate: 2020-10-23

Wu QW, JP Kapfhammer (2020)

The CRISPR-Cas13a system interferes with Purkinje cell dendritic development.

Biochimica et biophysica acta. Molecular cell research, 1867(7):118710.

RevDate: 2020-10-23
CmpDate: 2020-10-23

Ota A, Ishihara T, N Ishihara (2020)

Mitochondrial nucleoid morphology and respiratory function are altered in Drp1-deficient HeLa cells.

Journal of biochemistry, 167(3):287-294.

Mitochondria are dynamic organelles that frequently divide and fuse with each other. The dynamin-related GTPase protein Drp1 has a key role in mitochondrial fission. To analyse the physiological roles of Drp1 in cultured human cells, we analysed Drp1-deficient HeLa cells established by genome editing using CRISPR/Cas9. Under fluorescent microscopy, not only mitochondria were elongated but their DNA (mtDNA) nucleoids were extremely enlarged in bulb-like mitochondrial structures ('mito-bulbs') in the Drp1-deficient HeLa cells. We further found that respiratory activity, as measured by oxygen consumption rates, was severely repressed in Drp1-deficient HeLa cells and that this was reversible by the co-repression of mitochondrial fusion factors. Although mtDNA copy number was not affected, several respiratory subunits were repressed in Drp1-deficient HeLa cells. These results suggest that mitochondrial fission is required for the maintenance of active respiratory activity and the morphology of mtDNA nucleoids in human cells.

RevDate: 2020-10-23
CmpDate: 2020-10-23

Liu Q, Liu H, Li L, et al (2020)

ATAD2 predicts poor outcomes in patients with ovarian cancer and is a marker of proliferation.

International journal of oncology, 56(1):219-231.

The oncogene ATPase family AAA domain‑​containing protein 2 (ATAD2) has been demonstrated to promote malignancy in a number of different types of tumor; however, its expression and role in ovarian cancer (OC) remain unknown. In the present study, it was demonstrated that ATAD2 acts as both a marker and a driver of cell proliferation in OC. Immunohistochemistry (IHC) and bioinformatics analyses were used to evaluate ATAD2 expression in OC, and multi‑omics integrated analyses were used to dissect which factor resulted in its upregulation. Multiplex IHC assay was used to reveal the specific expression of ATAD2 in proliferating OC cells. CRISPR‑Cas9‑mediated gene editing was performed to investigate the effect of ATAD2 deletion on OC proliferation. The results demonstrated that ATAD2 is elevated in primary OC tissues compared with the adjacent normal tissue and metastases from the stomach. Genetic copy number amplification is a primary cause resulting in upregulation of ATAD2, and this was most frequently observed in OC. High ATAD2 expression was associated with advanced progression and predicted an unfavorable prognosis. ATAD2 could be used to identify cases of OC with a high proliferation signature and could label proliferating cells in OC. CRISPR‑Cas9‑mediated ATAD2 deletion resulted in a significant decrease in both cell proliferation and colony formation ability. Mechanistically, ATAD2‑knockdown resulted in deactivation of the mitogen‑activated protein kinase (MAPK) pathways, particularly the JNK‑MAPK pathway, resulting in suppression of proliferation. Collectively, the data from the present study demonstrated that the ATD2 gene was frequently amplified and protein expression levels were upregulated in OC. Therefore, ATAD2 may serve as an attractive diagnostic and prognostic OC marker, which may be used to identify patients with primary OC, whom are most likely to benefit from ATAD2 gene‑targeted proliferation intervention therapies.

RevDate: 2020-10-23
CmpDate: 2020-10-23

Bloomfield M, MC Louie (2019)

Chronic cadmium exposure decreases the dependency of MCF7 breast cancer cells on ERα.

Scientific reports, 9(1):12135 pii:10.1038/s41598-019-46912-3.

Cadmium is an environmental contaminant that can activate estrogen receptor alpha (ERα) and contribute to the development and progression of breast cancer. Our lab previously demonstrated that chronic cadmium exposure alters the expression of several ERα-responsive genes and increases the malignancy of breast cancer cells. Although these studies support cadmium's function as a hormone disrupter, the role of ERα in cadmium-induced breast cancer progression remains unclear. To address this, we modulated the expression of ERα and found that while the loss of ERα significantly impaired cancer cell growth, migration, invasion and anchorage-independent growth in both MCF7 and MCF7-Cd cells, cadmium-exposed cells retained a significant advantage in cell growth, migration, and invasion, and partially circumvented the loss of ERα. ERα knockout in MCF7 and MCF7-Cd cells significantly reduced the expression of classical ERα-regulated genes, while non-classical ERα-regulated genes were less impacted by the loss of ERα in MCF7-Cd cells. This is the first study to show that chronic cadmium exposure, even at low levels, can increase the malignancy of breast cancer cells by decreasing their dependency on ERα and increasing the adaptability of the cancer cells.

RevDate: 2020-10-23
CmpDate: 2020-10-23

Matsuda T, I Oinuma (2019)

Optimized CRISPR/Cas9-mediated in vivo genome engineering applicable to monitoring dynamics of endogenous proteins in the mouse neural tissues.

Scientific reports, 9(1):11309 pii:10.1038/s41598-019-47721-4.

To analyze the expression, localization, and functional dynamics of target proteins in situ, especially in living cells, it is important to develop a convenient, versatile, and efficient method to precisely introduce exogenous genes into the genome, which is applicable for labeling and engineering of the endogenous proteins of interest. By combining the CRISPR/Cas9 genome editing technology with an electroporation technique, we succeeded in creating knock-in alleles, from which GFP (RFP)-tagged endogenous proteins are produced, in neurons and glial cells in vivo in the developing mouse retina and brain. Correct gene targeting was confirmed by single-cell genotyping and Western blot analysis. Several gene loci were successfully targeted with high efficiency. Moreover, we succeeded in engineering the mouse genome to express foreign genes from the endogenous gene loci using a self-cleaving 2A peptide. Our method could be used to monitor the physiological changes in localization of endogenous proteins and expression levels of both mRNA and protein at a single cell resolution. This work discloses a powerful and widely applicable approach for visualization and manipulation of endogenous proteins in neural tissues.

RevDate: 2020-10-23
CmpDate: 2020-10-23

Roca Paixão JF, Gillet FX, Ribeiro TP, et al (2019)

Improved drought stress tolerance in Arabidopsis by CRISPR/dCas9 fusion with a Histone AcetylTransferase.

Scientific reports, 9(1):8080 pii:10.1038/s41598-019-44571-y.

Drought episodes decrease plant growth and productivity, which in turn cause high economic losses. Plants naturally sense and respond to water stress by activating specific signalling pathways leading to physiological and developmental adaptations. Genetically engineering genes that belong to these pathways might improve the drought tolerance of plants. The abscisic acid (ABA)-responsive element binding protein 1/ABRE binding factor (AREB1/ABF2) is a key positive regulator of the drought stress response. We investigated whether the CRISPR activation (CRISPRa) system that targets AREB1 might contribute to improve drought stress tolerance in Arabidopsis. Arabidopsis histone acetyltransferase 1 (AtHAT1) promotes gene expression activation by switching chromatin to a relaxed state. Stable transgenic plants expressing chimeric dCas9HAT were first generated. Then, we showed that the CRISPRa dCas9HAT mechanism increased the promoter activity controlling the β-glucuronidase (GUS) reporter gene. To activate the endogenous promoter of AREB1, the CRISPRa dCas9HAT system was set up, and resultant plants showed a dwarf phenotype. Our qRT-PCR experiments indicated that both AREB1 and RD29A, a gene positively regulated by AREB1, exhibited higher gene expression than the control plants. The plants generated here showed higher chlorophyll content and faster stomatal aperture under water deficit, in addition to a better survival rate after drought stress. Altogether, we report that CRISPRa dCas9HAT is a valuable biotechnological tool to improve drought stress tolerance through the positive regulation of AREB1.

RevDate: 2020-10-23
CmpDate: 2020-10-23

Yin Y, Reed EF, Q Zhang (2019)

Integrate CRISPR/Cas9 for protein expression of HLA-B*38:68Q via precise gene editing.

Scientific reports, 9(1):8067 pii:10.1038/s41598-019-44336-7.

The determination of null- or low-expressed HLA alleles is clinically relevant in both hematopoietic stem cell transplantation and solid organ transplantation. We studied the expression level of a questionable (Q) HLA-B*38:68Q allele, which carries a 9-nucleotide (nt) deletion at codon 230-232 in exon 4 of HLA-B*38:01:01:01 using CRISPR/Cas9 gene editing technology. CRISPR/Cas9 gene editing of HLA-B*38:01:01:01 homozygous EBV B cell line resulted in one HLA-B*38:68Q/B*38:01:01:01 heterozygous and one HLA-B*38:68Q homozygous clone. Flow cytometric analysis of monoclonal anti-Bw4 antibody showed the protein expression of HLA-B*38:01:01:01 in homozygous cells was 2.2 fold higher than HLA-B*38:68Q/B*38:01:01:01 heterozygous cells, and the expression of HLA-B*38:68Q/B*38:01:01:01 heterozygous cells was over 2.0 fold higher than HLA-B*38:68Q homozygous cells. The HLA-B*38:68Q expression was further confirmed using anti-B38 polyclonal antibody. Similarly, the expression of the HLA-B*38:01:01:01 homozygous cells was 1.5 fold higher than that of HLA-B*38:68Q/B*38:01:01:01 heterozygous cells, and the HLA-B*38:68Q/B*38:01:01:01 heterozygous cells was over 1.6 fold higher than that of HLA-B*38:68Q homozygous cells. The treatment of HLA-B*38:68Q homozygous cells with IFN-γ significantly increased its expression. In conclusion, we demonstrate that HLA-B*38:68Q is a low-expressing HLA allele. The CRISPR/Cas9 technology is a useful tool to induce precise gene editing in HLA genes to enable the characterization of HLA gene variants on expression and function.

RevDate: 2020-10-22

Lu Y, Tian Y, Shen R, et al (2020)

Precise Genome Modification in Tomato Using an Improved Prime Editing System.

Plant biotechnology journal [Epub ahead of print].

The CRISPR/Cas-mediated genome editing technology has been widely applied to create knock-out alleles of genes by generating short insertions or deletions (indel) in various plant species. Due to the low efficiency of homology-directed repair (HDR) and difficulties in the delivery of DNA template for HDR, precise genome editing remains challenging in plants (Mao et al., 2019). A tandem repeat-HDR method was developed very recently for sequence replacement in rice, which is most useful for monocots (Lu et al., 2020). Base editors developed from Cas9 nickase fusion with cytosine and adenine deaminases enable targeted C-to-T or A-to-G substitutions, but are restricted to specific types of base replacements and target site selections (Mao et al., 2019).

RevDate: 2020-10-22

Vats S, Bansal R, Rana N, et al (2020)

Unexplored nutritive potential of tomato to combat global malnutrition.

Critical reviews in food science and nutrition [Epub ahead of print].

Tomato, a widely consumed vegetable crop, offers a real potential to combat human nutritional deficiencies. Tomatoes are rich in micronutrients and other bioactive compounds (including vitamins, carotenoids, and minerals) that are known to be essential or beneficial for human health. This review highlights the current state of the art in the molecular understanding of the nutritional aspects, conventional and molecular breeding efforts, and biofortification studies undertaken to improve the nutritional content and quality of tomato. Transcriptomics and metabolomics studies, which offer a deeper understanding of the molecular regulation of the tomato's nutrients, are discussed. The potential uses of the wastes from the tomato processing industry (i.e., the peels and seed extracts) that are particularly rich in oils and proteins are also discussed. Recent advancements with CRISPR/Cas mediated gene-editing technology provide enormous opportunities to enhance the nutritional content of agricultural produces, including tomatoes. In this regard, genome editing efforts with respect to biofortification in the tomato plant are also discussed. The recent technological advancements and knowledge gaps described herein aim to help explore the unexplored nutritional potential of the tomato.

RevDate: 2020-10-22
CmpDate: 2020-10-22

Schwaller J (2020)

Learning from mouse models of MLL fusion gene-driven acute leukemia.

Biochimica et biophysica acta. Gene regulatory mechanisms, 1863(8):194550.

5-10% of human acute leukemias carry chromosomal translocations involving the mixed lineage leukemia (MLL) gene that result in the expression of chimeric protein fusing MLL to >80 different partners of which AF4, ENL and AF9 are the most prevalent. In contrast to many other leukemia-associated mutations, several MLL-fusions are powerful oncogenes that transform hematopoietic stem cells but also more committed progenitor cells. Here, I review different approaches that were used to express MLL fusions in the murine hematopoietic system which often, but not always, resulted in highly penetrant and transplantable leukemias that closely phenocopied the human disease. Due to its simple and reliable nature, reconstitution of irradiated mice with bone marrow cells retrovirally expressing the MLL-AF9 fusion became the most frequently in vivo model to study the biology of acute myeloid leukemia (AML). I review some of the most influential studies that used this model to dissect critical protein interactions, the impact of epigenetic regulators, microRNAs and microenvironment-dependent signals for MLL fusion-driven leukemia. In addition, I highlight studies that used this model for shRNA- or genome editing-based screens for cellular vulnerabilities that allowed to identify novel therapeutic targets of which some entered clinical trials. Finally, I discuss some inherent characteristics of the widely used mouse model based on retroviral expression of the MLL-AF9 fusion that can limit general conclusions for the biology of AML. This article is part of a Special Issue entitled: The MLL family of proteins in normal development and disease edited by Thomas A Milne.

RevDate: 2020-10-22
CmpDate: 2020-10-22

Zhang Y, Li H, Min YL, et al (2020)

Enhanced CRISPR-Cas9 correction of Duchenne muscular dystrophy in mice by a self-complementary AAV delivery system.

Science advances, 6(8):eaay6812.

Duchenne muscular dystrophy (DMD) is a lethal neuromuscular disease caused by mutations in the dystrophin gene (DMD). Previously, we applied CRISPR-Cas9-mediated "single-cut" genome editing to correct diverse genetic mutations in animal models of DMD. However, high doses of adeno-associated virus (AAV) are required for efficient in vivo genome editing, posing challenges for clinical application. In this study, we packaged Cas9 nuclease in single-stranded AAV (ssAAV) and CRISPR single guide RNAs in self-complementary AAV (scAAV) and delivered this dual AAV system into a mouse model of DMD. The dose of scAAV required for efficient genome editing were at least 20-fold lower than with ssAAV. Mice receiving systemic treatment showed restoration of dystrophin expression and improved muscle contractility. These findings show that the efficiency of CRISPR-Cas9-mediated genome editing can be substantially improved by using the scAAV system. This represents an important advancement toward therapeutic translation of genome editing for DMD.

RevDate: 2020-10-22
CmpDate: 2020-10-22

Hung IC, Chen TM, Lin JP, et al (2020)

Wnt5b integrates Fak1a to mediate gastrulation cell movements via Rac1 and Cdc42.

Open biology, 10(2):190273.

Focal adhesion kinase (FAK) mediates vital cellular pathways during development. Despite its necessity, how FAK regulates and integrates with other signals during early embryogenesis remains poorly understood. We found that the loss of Fak1a impaired epiboly, convergent extension and hypoblast cell migration in zebrafish embryos. We also observed a clear disturbance in cortical actin at the blastoderm margin and distribution of yolk syncytial nuclei. In addition, we investigated a possible link between Fak1a and a well-known gastrulation regulator, Wnt5b, and revealed that the overexpression of fak1a or wnt5b could cross-rescue convergence defects induced by a wnt5b or fak1a antisense morpholino (MO), respectively. Wnt5b and Fak1a were shown to converge in regulating Rac1 and Cdc42, which could synergistically rescue wnt5b and fak1a morphant phenotypes. Furthermore, we generated several alleles of fak1a mutants using CRISPR/Cas9, but those mutants only revealed mild gastrulation defects. However, injection of a subthreshold level of the wnt5b MO induced severe gastrulation defects in fak1a mutants, which suggested that the upregulated expression of wnt5b might complement the loss of Fak1a. Collectively, we demonstrated that a functional interaction between Wnt and FAK signalling mediates gastrulation cell movements via the possible regulation of Rac1 and Cdc42 and subsequent actin dynamics.

RevDate: 2020-10-22
CmpDate: 2020-10-22

Zhuang X, Veltri DP, EO Long (2019)

Genome-Wide CRISPR Screen Reveals Cancer Cell Resistance to NK Cells Induced by NK-Derived IFN-γ.

Frontiers in immunology, 10:2879.

The anti-leukemia activity of NK cells helps prevent relapse during hematopoietic stem cell transplantation (HSCT) in leukemia patients. However, the factors that determine the sensitivity or resistance of leukemia cells in the context of NK-mediated cytotoxicity are not well-established. Here, we performed a genome-wide CRISPR screen in the human chronic-myelogenous-leukemia (CML) cell line K562 to identify genes that regulate the vulnerability of leukemia cells to killing by primary human NK cells. The distribution of guide RNAs (gRNAs) in K562 cells that survived co-incubation with NK cells showed that loss of NCR3LG1, which encodes the ligand of the natural cytotoxicity receptor NKp30, protected K562 cells from killing. In contrast, loss of genes that regulate the antigen-presentation and interferon-γ-signaling pathways increased the vulnerability of K562 cells. The addition of IFN-γ neutralizing antibody increased the susceptibility of K562 cells to NK-mediated killing. Upregulation of MHC class I on K562 cells after co-incubation with NK cells was dependent on IFNGR2. Analysis of RNA-seq data from The Cancer Genome Atlas (TCGA) showed that low IFNGR2 expression in cancer tissues was associated with improved overall survival in acute myeloid leukemia (AML) and Kidney Renal Clear Cell Carcinoma (KIRC) patients. Our results, showing that the upregulation of MHC class I by NK-derived IFN-γ leads to resistance to NK cytotoxicity, suggest that targeting IFN-γ responses might be a promising approach to enhance NK cell anti-cancer efficacy.

RevDate: 2020-10-21

Vink JNA, Brouns SJJ, J Hohlbein (2020)

Extracting Transition Rates in Particle Tracking Using Analytical Diffusion Distribution Analysis.

Biophysical journal pii:S0006-3495(20)30765-7 [Epub ahead of print].

Single-particle tracking is an important technique in the life sciences to understand the kinetics of biomolecules. The analysis of apparent diffusion coefficients in vivo, for example, enables researchers to determine whether biomolecules are moving alone, as part of a larger complex, or are bound to large cellular components such as the membrane or chromosomal DNA. A remaining challenge has been to retrieve quantitative kinetic models, especially for molecules that rapidly switch between different diffusional states. Here, we present analytical diffusion distribution analysis (anaDDA), a framework that allows for extracting transition rates from distributions of apparent diffusion coefficients calculated from short trajectories that feature less than 10 localizations per track. Under the assumption that the system is Markovian and diffusion is purely Brownian, we show that theoretically predicted distributions accurately match simulated distributions and that anaDDA outperforms existing methods to retrieve kinetics, especially in the fast regime of 0.1-10 transitions per imaging frame. AnaDDA does account for the effects of confinement and tracking window boundaries. Furthermore, we added the option to perform global fitting of data acquired at different frame times to allow complex models with multiple states to be fitted confidently. Previously, we have started to develop anaDDA to investigate the target search of CRISPR-Cas complexes. In this work, we have optimized the algorithms and reanalyzed experimental data of DNA polymerase I diffusing in live Escherichia coli. We found that long-lived DNA interaction by DNA polymerase are more abundant upon DNA damage, suggesting roles in DNA repair. We further revealed and quantified fast DNA probing interactions that last shorter than 10 ms. AnaDDA pushes the boundaries of the timescale of interactions that can be probed with single-particle tracking and is a mathematically rigorous framework that can be further expanded to extract detailed information about the behavior of biomolecules in living cells.

RevDate: 2020-10-21

Minguet EG (2020)

Ares-GT: Design of guide RNAs targeting multiple genes for CRISPR-Cas experiments.

PloS one, 15(10):e0241001 pii:PONE-D-20-08048.

Guide RNA design for CRISPR genome editing of gene families is a challenging task as usually good candidate sgRNAs are tagged with low scores precisely because they match several locations in the genome, thus time-consuming manual evaluation of targets is required. To address this issues, I have developed ARES-GT, a Python local command line tool compatible with any operative system. ARES-GT allows the selection of candidate sgRNAs that match multiple input query sequences, in addition of candidate sgRNAs that specifically match each query sequence. It also contemplates the use of unmapped contigs apart from complete genomes thus allowing the use of any genome provided by user and being able to handle intraspecies allelic variability and individual polymorphisms. ARES-GT is available at GitHub (https://github.com/eugomin/ARES-GT.git).

RevDate: 2020-10-21

Störtz F, P Minary (2020)

crisprSQL: a novel database platform for CRISPR/Cas off-target cleavage assays.

Nucleic acids research pii:5934413 [Epub ahead of print].

With ongoing development of the CRISPR/Cas programmable nuclease system, applications in the area of in vivo therapeutic gene editing are increasingly within reach. However, non-negligible off-target effects remain a major concern for clinical applications. Even though a multitude of off-target cleavage datasets have been published, a comprehensive, transparent overview tool has not yet been established. Here, we present crisprSQL (http://www.crisprsql.com), an interactive and bioinformatically enhanced collection of CRISPR/Cas9 off-target cleavage studies aimed at enriching the fields of cleavage profiling, gene editing safety analysis and transcriptomics. The current version of crisprSQL contains cleavage data from 144 guide RNAs on 25,632 guide-target pairs from human and rodent cell lines, with interaction-specific references to epigenetic markers and gene names. The first curated database of this standard, it promises to enhance safety quantification research, inform experiment design and fuel development of computational off-target prediction algorithms.

RevDate: 2020-10-21

Shang Z, Chan SY, Song Q, et al (2020)

The Strategies of Pathogen-Oriented Therapy on Circumventing Antimicrobial Resistance.

Research (Washington, D.C.), 2020:2016201.

The emerging antimicrobial resistance (AMR) poses serious threats to the global public health. Conventional antibiotics have been eclipsed in combating with drug-resistant bacteria. Moreover, the developing and deploying of novel antimicrobial drugs have trudged, as few new antibiotics are being developed over time and even fewer of them can hit the market. Alternative therapeutic strategies to resolve the AMR crisis are urgently required. Pathogen-oriented therapy (POT) springs up as a promising approach in circumventing antibiotic resistance. The tactic underling POT is applying antibacterial compounds or materials directly to infected regions to treat specific bacteria species or strains with goals of improving the drug efficacy and reducing nontargeting and the development of drug resistance. This review exemplifies recent trends in the development of POTs for circumventing AMR, including the adoption of antibiotic-antibiotic conjugates, antimicrobial peptides, therapeutic monoclonal antibodies, nanotechnologies, CRISPR-Cas systems, and microbiota modulations. Employing these alternative approaches alone or in combination shows promising advantages for addressing the growing clinical embarrassment of antibiotics in fighting drug-resistant bacteria.

RevDate: 2020-10-21

Montenarh M, C Götz (2020)

Protein kinase CK2 and ion channels (Review).

Biomedical reports, 13(6):55.

Protein kinase CK2 appears as a tetramer or higher molecular weight oligomer composed of catalytic CK2α, CK2α' subunits and non-catalytic regulatory CK2β subunits or as individual subunits. It is implicated in a variety of different regulatory processes, such as Akt signalling, splicing and DNA repair within eukaryotic cells. The present review evaluates the influence of CK2 on ion channels in the plasma membrane. CK2 phosphorylates platform proteins such as calmodulin and ankyrin G, which bind to channel proteins for a physiological transport to and positioning into the membrane. In addition, CK2 directly phosphorylates a variety of channel proteins directly to regulate opening and closing of the channels. Thus, modulation of CK2 activities by specific inhibitors, by siRNA technology or by CRISPR/Cas technology has an influence on intracellular ion concentrations and thereby on cellular signalling. The physiological regulation of the intracellular ion concentration is important for cell survival and correct intracellular signalling. Disturbance of this regulation results in a variety of different diseases including epilepsy, heart failure, cystic fibrosis and diabetes. Therefore, these effects should be considered when using CK2 inhibition as a treatment option for cancer.

RevDate: 2020-10-21

Pinci F, Gaidt MM, Jung C, et al (2020)

C-tag TNF: a reporter system to study TNF shedding.

The Journal of biological chemistry pii:RA120.015248 [Epub ahead of print].

TNF is a highly pro-inflammatory cytokine that contributes not only to the regulation of immune responses but also to the development of severe inflammatory diseases. TNF is synthesized as a transmembrane protein, which is further matured via proteolytic cleavage by metalloproteases such as ADAM17, a process known as shedding. At present, TNF is mainly detected by measuring the precursor or the mature cytokine of bulk cell populations by techniques such as ELISA or immunoblotting. However, these methods do not provide information on the exact timing and extent of TNF cleavage at single-cell resolution and they do not allow the live visualization of shedding events. Here, we generated C-tag TNF as a genetically encoded reporter to study TNF shedding at the single-cell level. The functionality of the C-tag TNF reporter is based on the exposure of a cryptic epitope on the C-terminus of the transmembrane portion of pro-TNF upon cleavage. In both denatured and non-denatured samples, this epitope can be detected by a nanobody in a highly sensitive and specific manner only upon TNF shedding. As such, C-tag TNF can successfully be used for the detection of TNF cleavage in flow cytometry and live-cell imaging applications. We furthermore demonstrate its applicability in a forward genetic screen geared toward the identification of genetic regulators of TNF maturation. In summary, the C-tag TNF reporter can be employed to gain novel insights into the complex regulation of ADAM-dependent TNF shedding.

RevDate: 2020-10-21

Fernandez-Garcia L, Pacios O, González-Bardanca M, et al (2020)

Viral Related Tools against SARS-CoV-2.

Viruses, 12(10): pii:v12101172.

At the end of 2019, a new disease appeared and spread all over the world, the COVID-19, produced by the coronavirus SARS-CoV-2. As a consequence of this worldwide health crisis, the scientific community began to redirect their knowledge and resources to fight against it. Here we summarize the recent research on viruses employed as therapy and diagnostic of COVID-19: (i) viral-vector vaccines both in clinical trials and pre-clinical phases; (ii) the use of bacteriophages to find antibodies specific to this virus and some studies of how to use the bacteriophages themselves as a treatment against viral diseases; and finally, (iii) the use of CRISPR-Cas technology both to obtain a fast precise diagnose of the patient and also the possible use of this technology as a cure.

RevDate: 2020-10-21
CmpDate: 2020-10-21

Yang CH, Li HC, Ku TS, et al (2020)

MicroRNA-Independent Modulation of DICER1 Expression by hAgo2.

Molecular and cellular biology, 40(20):.

Many proteins, including DICER1 and hAgo2, are involved in the biogenesis of microRNAs (miRNAs). Whether hAgo2 regulates DICER1 expression is unknown. Exogenously overexpressed hAgo2 suppressed DICER1 expression at the levels of both protein and mRNA, and the reduction in hAgo2 expression enhanced DICER1 expression. Precursor miRNA processing mediated by DICER1 was also modulated by hAgo2. However, hAgo2 protein did not suppress DICER1 promoter activity. Therefore, hAgo2 protein probably regulates DICER1 expression at the posttranscriptional level. Indeed, hAgo2 protein inhibited the reporter assay of the DICER1 mRNA 3' untranslated region (3'-UTR). Previous reports have demonstrated that miRNAs (e.g., let-7 and miR-103/107) inhibited DICER1 expression posttranscriptionally. However, hAgo2 still suppressed DICER1 expression in the cells depleted of these miRNAs. Moreover, the reporter activities of the DICER1 mRNA 3'-UTR without these miRNA binding sites were still suppressed by hAgo2. Therefore, in addition to an miRNA-dependent pathway, hAgo2 can also modulate DICER1 expression through an miRNA-independent mechanism. Downregulation of DICER1 expression was further proven to be dependent on both hAgo2 and AUF1 proteins. Interactions of hAgo2 and AUF1 proteins were demonstrated by the coimmunoprecipitation assay. As expected, hAgo2 could not suppress the DICER1 mRNA 3'-UTR reporter with a mutation in the potential AUF1-binding site. Thus, downregulation of DICER1 expression through the 3'-UTR requires both hAgo2 and AUF1.

RevDate: 2020-10-21
CmpDate: 2020-10-21

Wilson AM, BD Wingfield (2020)

CRISPR-Cas9-Mediated Genome Editing in the Filamentous Ascomycete Huntiella omanensis.

Journal of visualized experiments : JoVE.

The CRISPR-Cas9 genome editing system is a molecular tool that can be used to introduce precise changes into the genomes of model and non-model species alike. This technology can be used for a variety of genome editing approaches, from gene knockouts and knockins to more specific changes like the introduction of a few nucleotides at a targeted location. Genome editing can be used for a multitude of applications, including the partial functional characterization of genes, the production of transgenic organisms and the development of diagnostic tools. Compared to previously available gene editing strategies, the CRISPR-Cas9 system has been shown to be easy to establish in new species and boasts high efficiency and specificity. The primary reason for this is that the editing tool uses an RNA molecule to target the gene or sequence of interest, making target molecule design straightforward, given that standard base pairing rules can be exploited. Similar to other genome editing systems, CRISPR-Cas9-based methods also require efficient and effective transformation protocols as well as access to good quality sequence data for the design of the targeting RNA and DNA molecules. Since the introduction of this system in 2013, it has been used to genetically engineer a variety of model species, including Saccharomyces cerevisiae, Arabidopsis thaliana, Drosophila melanogaster and Mus musculus. Subsequently, researchers working on non-model species have taken advantage of the system and used it for the study of genes involved in processes as diverse as secondary metabolism in fungi, nematode growth and disease resistance in plants, among many others. This protocol detailed below describes the use of the CRISPR-Cas9 genome editing protocol for the truncation of a gene involved in the sexual cycle of Huntiella omanensis, a filamentous ascomycete fungus belonging to the Ceratocystidaceae family.

RevDate: 2020-10-21
CmpDate: 2020-10-21

Kalwani P, Rath D, A Ballal (2020)

Novel molecular aspects of the CRISPR backbone protein 'Cas7' from cyanobacteria.

The Biochemical journal, 477(5):971-983.

The cyanobacterium Anabaena PCC 7120 shows the presence of Type I-D CRISPR system that can potentially confer adaptive immunity. The Cas7 protein (Alr1562), which forms the backbone of the type I-D surveillance complex, was characterized from Anabaena. Alr1562, showed the presence of the non-canonical RNA recognition motif and two intrinsically disordered regions (IDRs). When overexpressed in E. coli, the Alr1562 protein was soluble and could be purified by affinity chromatography, however, deletion of IDRs rendered Alr1562 completely insoluble. The purified Alr1562 was present in the dimeric or a RNA-associated higher oligomeric form, which appeared as spiral structures under electron microscope. With RNaseA and NaCl treatment, the higher oligomeric form converted to the lower oligomeric form, indicating that oligomerization occurred due to the association of Alr1562 with RNA. The secondary structure of both these forms was largely similar, resembling that of a partially folded protein. The dimeric Alr1562 was more prone to temperature-dependent aggregation than the higher oligomeric form. In vitro, the Alr1562 bound more specifically to a minimal CRISPR unit than to the non-specific RNA. Residues required for binding of Alr1562 to RNA, identified by protein modeling-based approaches, were mutated for functional validation. Interestingly, these mutant proteins, showing reduced ability to bind RNA were predominantly present in dimeric form. Alr1562 was detected with specific antiserum in Anabaena, suggesting that the type I-D system is expressed and may be functional in vivo. This is the first report that describes the characterization of a Cas protein from any photosynthetic organism.

RevDate: 2020-10-21
CmpDate: 2020-10-21

Diaz-de-la-Loza MD, Loker R, Mann RS, et al (2020)

Control of tissue morphogenesis by the HOX gene Ultrabithorax.

Development (Cambridge, England), 147(5):.

Mutations in the Ultrabithorax (Ubx) gene cause homeotic transformation of the normally two-winged Drosophila into a four-winged mutant fly. Ubx encodes a HOX family transcription factor that specifies segment identity, including transformation of the second set of wings into rudimentary halteres. Ubx is known to control the expression of many genes that regulate tissue growth and patterning, but how it regulates tissue morphogenesis to reshape the wing into a haltere is still unclear. Here, we show that Ubx acts by repressing the expression of two genes in the haltere, Stubble and Notopleural, both of which encode transmembrane proteases that remodel the apical extracellular matrix to promote wing morphogenesis. In addition, Ubx induces expression of the Tissue inhibitor of metalloproteases in the haltere, which prevents the basal extracellular matrix remodelling necessary for wing morphogenesis. Our results provide a long-awaited explanation for how Ubx controls morphogenetic transformation.

RevDate: 2020-10-21
CmpDate: 2020-10-21

Raja DA, Subramaniam Y, Aggarwal A, et al (2020)

Histone variant dictates fate biasing of neural crest cells to melanocyte lineage.

Development (Cambridge, England), 147(5): pii:dev.182576.

In the neural crest lineage, progressive fate restriction and stem cell assignment are crucial for both development and regeneration. Whereas fate commitment events have distinct transcriptional footprints, fate biasing is often transitory and metastable, and is thought to be moulded by epigenetic programmes. Therefore, the molecular basis of specification is difficult to define. In this study, we established a role for a histone variant, H2a.z.2, in specification of the melanocyte lineage from multipotent neural crest cells. H2a.z.2 silencing reduces the number of melanocyte precursors in developing zebrafish embryos and from mouse embryonic stem cells in vitro We demonstrate that this histone variant occupies nucleosomes in the promoter of the key melanocyte determinant mitf, and enhances its induction. CRISPR/Cas9-based targeted mutagenesis of this gene in zebrafish drastically reduces adult melanocytes, as well as their regeneration. Thereby, our study establishes the role of a histone variant upstream of the core gene regulatory network in the neural crest lineage. This epigenetic mark is a key determinant of cell fate and facilitates gene activation by external instructive signals, thereby establishing melanocyte fate identity.

RevDate: 2020-10-21
CmpDate: 2020-10-21

Schulte LN, Schweinlin M, Westermann AJ, et al (2020)

An Advanced Human Intestinal Coculture Model Reveals Compartmentalized Host and Pathogen Strategies during Salmonella Infection.

mBio, 11(1):.

A major obstacle in infection biology is the limited ability to recapitulate human disease trajectories in traditional cell culture and animal models, which impedes the translation of basic research into clinics. Here, we introduce a three-dimensional (3D) intestinal tissue model to study human enteric infections at a level of detail that is not achieved by conventional two-dimensional monocultures. Our model comprises epithelial and endothelial layers, a primary intestinal collagen scaffold, and immune cells. Upon Salmonella infection, the model mimics human gastroenteritis, in that it restricts the pathogen to the epithelial compartment, an advantage over existing mouse models. Application of dual transcriptome sequencing to the Salmonella-infected model revealed the communication of epithelial, endothelial, monocytic, and natural killer cells among each other and with the pathogen. Our results suggest that Salmonella uses its type III secretion systems to manipulate STAT3-dependent inflammatory responses locally in the epithelium without accompanying alterations in the endothelial compartment. Our approach promises to reveal further human-specific infection strategies employed by Salmonella and other pathogens.IMPORTANCE Infection research routinely employs in vitro cell cultures or in vivo mouse models as surrogates of human hosts. Differences between murine and human immunity and the low level of complexity of traditional cell cultures, however, highlight the demand for alternative models that combine the in vivo-like properties of the human system with straightforward experimental perturbation. Here, we introduce a 3D tissue model comprising multiple cell types of the human intestinal barrier, a primary site of pathogen attack. During infection with the foodborne pathogen Salmonella enterica serovar Typhimurium, our model recapitulates human disease aspects, including pathogen restriction to the epithelial compartment, thereby deviating from the systemic infection in mice. Combination of our model with state-of-the-art genetics revealed Salmonella-mediated local manipulations of human immune responses, likely contributing to the establishment of the pathogen's infection niche. We propose the adoption of similar 3D tissue models to infection biology, to advance our understanding of molecular infection strategies employed by bacterial pathogens in their human host.

RevDate: 2020-10-21
CmpDate: 2020-10-21

Duong T, Rasmussen NR, Ballato E, et al (2020)

The Rheb-TORC1 signaling axis functions as a developmental checkpoint.

Development (Cambridge, England), 147(5):.

In many eukaryotes, the small GTPase Rheb functions as a switch to toggle activity of TOR complex 1 (TORC1) between anabolism and catabolism, thus controlling lifespan, development and autophagy. Our CRISPR-generated, fluorescently tagged endogenous Caenorhabditis elegans RHEB-1 and DAF-15/Raptor are expressed ubiquitously and localize to lysosomes. LET-363/TOR and DAF-15/Raptor are required for development beyond the third larval stage (L3). We observed that deletion of RHEB-1 similarly conferred L3 arrest. Unexpectedly, robust RNAi-mediated depletion of TORC1 components caused arrest at stages prior to L3. Accordingly, conditional depletion of endogenous DAF-15/Raptor in the soma revealed that TORC1 is required at each stage of the life cycle to progress to the next stage. Reversal of DAF-15 depletion permits arrested animals to recover to continue development. Our results are consistent with TORC1 functioning as a developmental checkpoint that governs the decision of the animal to progress through development.

RevDate: 2020-10-21
CmpDate: 2020-10-21

Dai Y, Somoza RA, Wang L, et al (2019)

Exploring the Trans-Cleavage Activity of CRISPR-Cas12a (cpf1) for the Development of a Universal Electrochemical Biosensor.

Angewandte Chemie (International ed. in English), 58(48):17399-17405.

An accurate, rapid, and cost-effective biosensor for the quantification of disease biomarkers is vital for the development of early-diagnostic point-of-care systems. The recent discovery of the trans-cleavage property of CRISPR type V effectors makes CRISPR a potential high-accuracy bio-recognition tool. Herein, a CRISPR-Cas12a (cpf1) based electrochemical biosensor (E-CRISPR) is reported, which is more cost-effective and portable than optical-transduction-based biosensors. Through optimizing the in vitro trans-cleavage activity of Cas12a, E-CRIPSR was used to detect viral nucleic acids, including human papillomavirus 16 (HPV-16) and parvovirus B19 (PB-19), with a picomolar sensitivity. An aptamer-based E-CRISPR cascade was further designed for the detection of transforming growth factor β1 (TGF-β1) protein in clinical samples. As demonstrated, E-CRISPR could enable the development of portable, accurate, and cost-effective point-of-care diagnostic systems.

RevDate: 2020-10-20

Lenskaia T, D Boley (2020)

Prokaryote autoimmunity in the context of self-targeting by CRISPR-Cas systems.

Journal of bioinformatics and computational biology [Epub ahead of print].

Prokaryote adaptive immunity (CRISPR-Cas systems) can be a threat to its carriers. We analyze the risks of autoimmune reactions related to adaptive immunity in prokaryotes by computational methods. We found important differences between bacteria and archaea with respect to autoimmunity potential. According to the results of our analysis, CRISPR-Cas systems in bacteria are more prone to self-targeting even though they possess fewer spacers per organism on average than archaea. The results of our study provide opportunities to use self-targeting in prokaryotes for biological and medical applications.

RevDate: 2020-10-20

Csörgő B, León LM, Chau-Ly IJ, et al (2020)

A compact Cascade-Cas3 system for targeted genome engineering.

Nature methods pii:10.1038/s41592-020-00980-w [Epub ahead of print].

CRISPR-Cas technologies have enabled programmable gene editing in eukaryotes and prokaryotes. However, the leading Cas9 and Cas12a enzymes are limited in their ability to make large deletions. Here, we used the processive nuclease Cas3, together with a minimal Type I-C Cascade-based system for targeted genome engineering in bacteria. DNA cleavage guided by a single CRISPR RNA generated large deletions (7-424 kilobases) in Pseudomonas aeruginosa with near-100% efficiency, while Cas9 yielded small deletions and point mutations. Cas3 generated bidirectional deletions originating from the programmed site, which was exploited to reduce the P. aeruginosa genome by 837 kb (13.5%). Large deletion boundaries were efficiently specified by a homology-directed repair template during editing with Cascade-Cas3, but not Cas9. A transferable 'all-in-one' vector was functional in Escherichia coli, Pseudomonas syringae and Klebsiella pneumoniae, and endogenous CRISPR-Cas use was enhanced with an 'anti-anti-CRISPR' strategy. P. aeruginosa Type I-C Cascade-Cas3 (PaeCas3c) facilitates rapid strain manipulation with applications in synthetic biology, genome minimization and the removal of large genomic regions.

RevDate: 2020-10-20

Pilosof S, Alcalá-Corona SA, Wang T, et al (2020)

The network structure and eco-evolutionary dynamics of CRISPR-induced immune diversification.

Nature ecology & evolution pii:10.1038/s41559-020-01312-z [Epub ahead of print].

As a heritable sequence-specific adaptive immune system, CRISPR-Cas is a powerful force shaping strain diversity in host-virus systems. While the diversity of CRISPR alleles has been explored, the associated structure and dynamics of host-virus interactions have not. We explore the role of CRISPR in mediating the interplay between host-virus interaction structure and eco-evolutionary dynamics in a computational model and compare the results with three empirical datasets from natural systems. We show that the structure of the networks describing who infects whom and the degree to which strains are immune, are respectively modular (containing groups of hosts and viruses that interact strongly) and weighted-nested (specialist hosts are more susceptible to subsets of viruses that in turn also infect the more generalist hosts with many spacers matching many viruses). The dynamic interplay between these networks influences transitions between dynamical regimes of virus diversification and host control. The three empirical systems exhibit weighted-nested immunity networks, a pattern our theory shows is indicative of hosts able to suppress virus diversification. Previously missing from studies of microbial host-pathogen systems, the immunity network plays a key role in the coevolutionary dynamics.

RevDate: 2020-10-20

Jin S, Bae J, Song Y, et al (2020)

Synthetic Biology on Acetogenic Bacteria for Highly Efficient Conversion of C1 Gases to Biochemicals.

International journal of molecular sciences, 21(20): pii:ijms21207639.

Synthesis gas, which is mainly produced from fossil fuels or biomass gasification, consists of C1 gases such as carbon monoxide, carbon dioxide, and methane as well as hydrogen. Acetogenic bacteria (acetogens) have emerged as an alternative solution to recycle C1 gases by converting them into value-added biochemicals using the Wood-Ljungdahl pathway. Despite the advantage of utilizing acetogens as biocatalysts, it is difficult to develop industrial-scale bioprocesses because of their slow growth rates and low productivities. To solve these problems, conventional approaches to metabolic engineering have been applied; however, there are several limitations owing to the lack of required genetic bioparts for regulating their metabolic pathways. Recently, synthetic biology based on genetic parts, modules, and circuit design has been actively exploited to overcome the limitations in acetogen engineering. This review covers synthetic biology applications to design and build industrial platform acetogens.

RevDate: 2020-10-19

Tran MT, Doan DTH, Kim J, et al (2020)

CRISPR/Cas9-based precise excision of SlHyPRP1 domain(s) to obtain salt stress-tolerant tomato.

Plant cell reports pii:10.1007/s00299-020-02622-z [Epub ahead of print].

KEY MESSAGE: CRISPR/Cas9-based multiplexed editing of SlHyPRP1 resulted in precise deletions of its functional motif(s), thereby resulting in salt stress-tolerant events in cultivated tomato. Crop genetic improvement to address environmental stresses for sustainable food production has been in high demand, especially given the current situation of global climate changes and reduction of the global food production rate/population rate. Recently, the emerging clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas)-based targeted mutagenesis has provided a revolutionary approach to crop improvement. The major application of CRISPR/Cas in plant genome editing has been the generation of indel mutations via error-prone nonhomologous end joining (NHEJ) repair of DNA DSBs. In this study, we examined the power of the CRISPR/Cas9-based novel approach in the precise manipulation of protein domains of tomato hybrid proline-rich protein 1 (HyPRP1), which is a negative regulator of salt stress responses. We revealed that the precise elimination of SlHyPRP1 negative-response domain(s) led to high salinity tolerance at the germination and vegetative stages in our experimental conditions. CRISPR/Cas9-based domain editing may be an efficient tool to engineer multidomain proteins of important food crops to cope with global climate changes for sustainable agriculture and future food security.

RevDate: 2020-10-19

Dhokane D, Bhadra B, S Dasgupta (2020)

CRISPR based targeted genome editing of Chlamydomonas reinhardtii using programmed Cas9-gRNA ribonucleoprotein.

Molecular biology reports pii:10.1007/s11033-020-05922-5 [Epub ahead of print].

The clustered regularly interspaced short palindromic repeats (CRISPR) - Cas associated protein 9 (Cas9) system is very precise, efficient and relatively simple in creating genetic modifications at a predetermined locus in the genome. Genome editing with Cas9 ribonucleoproteins (RNPs) has reduced cytotoxic effects, off-target cleavage and increased on-target activity and the editing efficiencies. The unicellular alga Chlamydomonas reinhardtii is an emerging model for studying the production of high-value products for industrial applications. Development of C. reinhardtii as an industrial biotechnology host can be achieved more efficiently through genetic modifications using genome editing tools. We made an attempt to target MAA7 gene that encodes the tryptophan synthase β-Subunit using CRISPR-Cas9 RNPs to demonstrate knock-out and knock-in through homology-dependent repair template at the target site. In this study, we have demonstrated targeted gene knock-out in C. reinhardtii using programmed RNPs. Targeted editing of MAA7 gene was confirmed by sequencing the clones that were resistant to 5-Fluoroindole (5-FI). Non-homologous end joining (NHEJ) repair mechanism led to insertion, deletion, and/or base substitution in the Cas9 cleavage vicinity, encoding non-functional MAA7 protein product (knock-out), conferring resistance to 5-FI. Here, we report an efficient protocol for developing knock-out mutants in Chlamydomonas using CRISPR-Cas9 RNPs. The high potential efficiency of editing may also eliminate the need to select mutants by phenotype. These research findings would be more likely applied to other green algae for developing green cell factories to produce high-value molecules.

RevDate: 2020-10-19

Hampton HG, Smith LM, Ferguson S, et al (2020)

Functional genomics reveals the toxin-antitoxin repertoire and AbiE activity in Serratia.

Microbial genomics [Epub ahead of print].

Bacteriophage defences are divided into innate and adaptive systems. Serratia sp. ATCC 39006 has three CRISPR-Cas adaptive immune systems, but its innate immune repertoire is unknown. Here, we re-sequenced and annotated the Serratia genome and predicted its toxin-antitoxin (TA) systems. TA systems can provide innate phage defence through abortive infection by causing infected cells to 'shut down', limiting phage propagation. To assess TA system function on a genome-wide scale, we utilized transposon insertion and RNA sequencing. Of the 32 TA systems predicted bioinformatically, 4 resembled pseudogenes and 11 were demonstrated to be functional based on transposon mutagenesis. Three functional systems belonged to the poorly characterized but widespread, AbiE, abortive infection/TA family. AbiE is a type IV TA system with a predicted nucleotidyltransferase toxin. To investigate the mode of action of this toxin, we measured the transcriptional response to AbiEii expression. We observed dysregulated levels of tRNAs and propose that the toxin targets tRNAs resulting in bacteriostasis. A recent report on a related toxin shows this occurs through addition of nucleotides to tRNA(s). This study has demonstrated the utility of functional genomics for probing TA function in a high-throughput manner, defined the TA repertoire in Serratia and shown the consequences of AbiE induction.

RevDate: 2020-10-20
CmpDate: 2020-10-20

Martinez-Lage M, Torres-Ruiz R, Puig-Serra P, et al (2020)

In vivo CRISPR/Cas9 targeting of fusion oncogenes for selective elimination of cancer cells.

Nature communications, 11(1):5060.

Fusion oncogenes (FOs) are common in many cancer types and are powerful drivers of tumor development. Because their expression is exclusive to cancer cells and their elimination induces cell apoptosis in FO-driven cancers, FOs are attractive therapeutic targets. However, specifically targeting the resulting chimeric products is challenging. Based on CRISPR/Cas9 technology, here we devise a simple, efficient and non-patient-specific gene-editing strategy through targeting of two introns of the genes involved in the rearrangement, allowing for robust disruption of the FO specifically in cancer cells. As a proof-of-concept of its potential, we demonstrate the efficacy of intron-based targeting of transcription factors or tyrosine kinase FOs in reducing tumor burden/mortality in in vivo models. The FO targeting approach presented here might open new horizons for the selective elimination of cancer cells.

RevDate: 2020-10-19
CmpDate: 2020-10-19

Chen Y, Shi Y, Chen Y, et al (2020)

Contamination-free visual detection of SARS-CoV-2 with CRISPR/Cas12a: A promising method in the point-of-care detection.

Biosensors & bioelectronics, 169:112642.

The outbreaks of the infectious disease COVID-19 caused by SARS-CoV-2 seriously threatened the life of humans. A rapid, reliable and specific detection method was urgently needed. Herein, we reported a contamination-free visual detection method for SARS-CoV-2 with LAMP and CRISPR/Cas12a technology. CRISPR/Cas12a reagents were pre-added on the inner wall of the tube lid. After LAMP reaction, CRISPR/Cas12a reagents were flowed into the tube and mixed with amplicon solution by hand shaking, which can effectively avoid possible amplicon formed aerosol contamination caused by re-opening the lid after amplification. CRISPR/Cas12a can highly specific recognize target sequence and discriminately cleave single strand DNA probes (5'-6FAM 3'-BHQ1). With smart phone and portable 3D printing instrument, the produced fluorescence can be seen by naked eyes without any dedicated instruments, which is promising in the point-of-care detection. The whole amplification and detection process could be completed within 40 min with high sensitivity of 20 copies RNA of SARS-CoV-2. This reaction had high specificity and could avoid cross-reactivity with other common viruses such as influenza virus. For 7 positive and 3 negative respiratory swab samples provided by Zhejiang Provincial Center for Disease Control and Prevention, our detection results had 100% positive agreement and 100% negative agreement, which demonstrated the accuracy and application prospect of this method.

RevDate: 2020-10-19
CmpDate: 2020-10-19

Zou Y, Henry WS, Ricq EL, et al (2020)

Plasticity of ether lipids promotes ferroptosis susceptibility and evasion.

Nature, 585(7826):603-608.

Ferroptosis-an iron-dependent, non-apoptotic cell death process-is involved in various degenerative diseases and represents a targetable susceptibility in certain cancers1. The ferroptosis-susceptible cell state can either pre-exist in cells that arise from certain lineages or be acquired during cell-state transitions2-5. However, precisely how susceptibility to ferroptosis is dynamically regulated remains poorly understood. Here we use genome-wide CRISPR-Cas9 suppressor screens to identify the oxidative organelles peroxisomes as critical contributors to ferroptosis sensitivity in human renal and ovarian carcinoma cells. Using lipidomic profiling we show that peroxisomes contribute to ferroptosis by synthesizing polyunsaturated ether phospholipids (PUFA-ePLs), which act as substrates for lipid peroxidation that, in turn, results in the induction of ferroptosis. Carcinoma cells that are initially sensitive to ferroptosis can switch to a ferroptosis-resistant state in vivo in mice, which is associated with extensive downregulation of PUFA-ePLs. We further find that the pro-ferroptotic role of PUFA-ePLs can be extended beyond neoplastic cells to other cell types, including neurons and cardiomyocytes. Together, our work reveals roles for the peroxisome-ether-phospholipid axis in driving susceptibility to and evasion from ferroptosis, highlights PUFA-ePL as a distinct functional lipid class that is dynamically regulated during cell-state transitions, and suggests multiple regulatory nodes for therapeutic interventions in diseases that involve ferroptosis.

RevDate: 2020-10-19
CmpDate: 2020-10-19

Joung J, Ladha A, Saito M, et al (2020)

Detection of SARS-CoV-2 with SHERLOCK One-Pot Testing.

The New England journal of medicine, 383(15):1492-1494.

RevDate: 2020-10-20
CmpDate: 2020-10-20

Fu R, Fang M, Xu K, et al (2020)

Generation of GGTA1-/-β2M-/-CIITA-/- Pigs Using CRISPR/Cas9 Technology to Alleviate Xenogeneic Immune Reactions.

Transplantation, 104(8):1566-1573.

BACKGROUND: Xenogeneic organ transplantation has been proposed as a potential approach to fundamentally solve organ shortage problem. Xenogeneic immune responses across species is one of the major obstacles for clinic application of xeno-organ transplantation. The generation of glycoprotein galactosyltransferase α 1, 3 (GGTA1) knockout pigs has greatly contributed to the reduction of hyperacute xenograft rejection. However, severe xenograft rejection can still be induced by xenoimmune responses to the porcine major histocompatibility complex antigens swine leukocyte antigen class I and class II.

METHODS: We simultaneously depleted GGTA1, β2-microglobulin (β2M), and major histocompatibility complex class II transactivator (CIITA) genes using clustered regularly interspaced short palindromic repeats and CRISPR-associated proteins technology in Bamma pig fibroblast cells, which were further used to generate GGTA1β2MCIITA triple knockout (GBC-3KO) pigs by nuclear transfer.

RESULTS: The genotype of GBC-3KO pigs was confirmed by polymerase chain reaction and Sanger sequencing, and the loss of expression of α-1,3-galactose, SLA-I, and SLA-II was demonstrated by flow cytometric analysis using fluorescent-conjugated lectin from bandeiraea simplicifolia, anti-β2-microglobulin, and swine leukocyte antigen class II DR antibodies. Furthermore, mixed lymphocyte reaction assay revealed that peripheral blood mononuclear cells from GBC-3KO pigs were significantly less effective than (WT) pig peripheral blood mononuclear cells in inducing human CD3CD4 and CD3CD8 T-cell activation and proliferation. In addition, GBC-3KO pig skin grafts showed a significantly prolonged survival in immunocompetent C57BL/6 mice, when compared with wild-type pig skin grafts.

CONCLUSIONS: Taken together, these results demonstrate that elimination of GGTA1, β2M, and CIITA genes in pigs can effectively alleviate xenogeneic immune responses and prolong pig organ survival in xenogenesis. We believe that this work will facilitate future research in xenotransplantation.

RevDate: 2020-10-19
CmpDate: 2020-10-19

Tang X, Suo J, Liang L, et al (2020)

Genetic modification of the protozoan Eimeria tenella using the CRISPR/Cas9 system.

Veterinary research, 51(1):41.

Eimeria tenella has emerged as valuable model organism for studying the biology and immunology of protozoan parasites with the establishment of the reverse genetic manipulation platform. In this report, we described the application of CRISPR (clustered regularly interspaced short palindromic repeat)/Cas9 (endonuclease) system for efficient genetic editing in E. tenella, and showed that the CRISPR/Cas9 system mediates site-specific double-strand DNA breaks with a single guide RNA. Using this system, we successfully tagged the endogenous microneme protein 2 (EtMic2) by inserting the red fluorescent protein into the C-terminal of EtMic2. Our results extended the utility of the CRISPR/Cas9-mediated genetic modification system to E. tenella, and opened a new avenue for targeted investigation of gene functions in apicomplexan parasites.

RevDate: 2020-10-19
CmpDate: 2020-10-19

Tiraboschi E, Martina S, van der Ent W, et al (2020)

New insights into the early mechanisms of epileptogenesis in a zebrafish model of Dravet syndrome.

Epilepsia, 61(3):549-560.

OBJECTIVE: To pinpoint the earliest cellular defects underlying seizure onset (epileptogenic period) during perinatal brain development in a new zebrafish model of Dravet syndrome (DS) and to investigate potential disease-modifying activity of the 5HT2 receptor agonist fenfluramine.

METHODS: We used CRISPR/Cas9 mutagenesis to introduce a missense mutation, designed to perturb ion transport function in all channel isoforms, into scn1lab, the zebrafish orthologue of SCN1A (encoding voltage-gated sodium channel alpha subunit 1). We performed behavioral analysis and electroencephalographic recordings to measure convulsions and epileptiform discharges, followed by single-cell RNA-Seq, morphometric analysis of transgenic reporter-labeled γ-aminobutyric acidergic (GABAergic) neurons, and pharmacological profiling of mutant larvae.

RESULTS: Homozygous mutant (scn1labmut/mut) larvae displayed spontaneous seizures with interictal, preictal, and ictal discharges (mean = 7.5 per 20-minute recording; P < .0001; one-way analysis of variance). Drop-Seq analysis revealed a 2:1 shift in the ratio of glutamatergic to GABAergic neurons in scn1labmut/mut larval brains versus wild type (WT), with dynamic changes in neuronal, glial, and progenitor cell populations. To explore disease pathophysiology further, we quantified dendritic arborization in GABAergic neurons and observed a 40% reduction in arbor number compared to WT (P < .001; n = 15 mutant, n = 16 WT). We postulate that the significant reduction in inhibitory arbors causes an inhibitory to excitatory neurotransmitter imbalance that contributes to seizures and enhanced electrical brain activity in scn1labmut/mut larvae (high-frequency range), with subsequent GABAergic neuronal loss and astrogliosis. Chronic fenfluramine administration completely restored dendritic arbor numbers to normal in scn1labmut/mut larvae, whereas similar treatment with the benzodiazepine diazepam attenuated seizures, but was ineffective in restoring neuronal cytoarchitecture. BrdU labeling revealed cell overproliferation in scn1labmut/mut larval brains that were rescued by fenfluramine but not diazepam.

SIGNIFICANCE: Our findings provide novel insights into early mechanisms of DS pathogenesis, describe dynamic cell population changes in the scn1labmut/mut brain, and present first-time evidence for potential disease modification by fenfluramine.

RevDate: 2020-10-19
CmpDate: 2020-10-19

Gabel I, J Moreno (2019)

Genome Editing, Ethics, and Politics.

AMA journal of ethics, 21(12):E1105-1110.

For the better part of a dozen years and over 3 US presidential terms, heated debates about the ethics of cloning and embryonic stem cell research helped to define the American political landscape. Current lack of public controversy about regulation of human genome editing does not signal that ethical issues about engineering human embryos have been settled. Rather, while genome editing raises old ethical questions about the value of human life, eugenics, and the weight of unintended consequences, it also came into being in a political landscape that vastly differs from the early aughts when bioethics was last a major topic of political controversy.

RevDate: 2020-10-19
CmpDate: 2020-10-19

Blasimme A (2019)

Why Include the Public in Genome Editing Governance Deliberation?.

AMA journal of ethics, 21(12):E1065-1070 pii:amajethics.2019.1065.

With the birth of genetically engineered twins in November 2018, international debate about human genome editing governance has moved from an emphasis on mutual engagement among multiple stakeholders to a self-regulatory model enacted through high-level expert groups with little or no public input. This article reconstructs this paradigm shift and suggests that inclusive public deliberation should still have a role in public decision making about genome editing.

RevDate: 2020-10-19
CmpDate: 2020-10-19

Schweikart SJ (2019)

What Is Prudent Governance of Human Genome Editing?.

AMA journal of ethics, 21(12):E1042-1048 pii:amajethics.2019.1042.

CRISPR technology has made questions about how best to regulate human genome editing immediately relevant. A sound and ethical governance structure for human genome editing is necessary, as the consequences of this new technology are far-reaching and profound. Because there are currently many risks associated with genome editing technology, the extent of which are unknown, regulatory prudence is ideal. When considering how best to create a prudent governance scheme, we can look to 2 guiding examples: the Asilomar conference of 1975 and the German Ethics Council guidelines for human germline intervention. Both models offer a path towards prudent regulation in the face of unknown and significant risks.

RevDate: 2020-10-19
CmpDate: 2020-10-19

Thompson C (2019)

How Should "CRISPRed" Babies Be Monitored Over Their Life Course to Promote Health Equity?.

AMA journal of ethics, 21(12):E1036-1041 pii:amajethics.2019.1036.

Gene-edited babies who might be born in the future should be monitored over the course of their life. These patients' physical, mental, and social health monitoring should be coordinated by clinicians in ways that anonymize patients' data for privacy protection but also allow for national and international aggregate evaluations. Transnational monitoring efforts should focus on safety and efficacy, social and disability justice, what constitutes the standard of care, and how best to promote both access to care and social and genomic research and innovation. In addition, effective and binding mechanisms for stopping or limiting uses of gene editing technology should be developed.

RevDate: 2020-10-19
CmpDate: 2020-10-19

Lehmann LS (2019)

Using the 4-S Framework to Guide Conversations With Patients About CRISPR.

AMA journal of ethics, 21(12):E1029-1035 pii:amajethics.2019.1029.

As patients with genetic diseases seek to have healthy biologically connected children, they will undoubtedly turn to trusted health care professionals for guidance. "Doctor, should I enter a clinical trial to edit my embryos?" is likely to become a query posed by patients with genetic illnesses. Physicians need both empathic communication skills and a framework for responding to this question. Applying the 4-S framework to gene editing can guide clinicians' responses to patients' CRISPR queries by facilitating discussion of (1) safety, (2) significance of harm to be averted, (3) impact on succeeding generations, and (4) social consequences.

RevDate: 2020-10-19
CmpDate: 2020-10-19

Shohayeb B, Ho U, Yeap YY, et al (2020)

The association of microcephaly protein WDR62 with CPAP/IFT88 is required for cilia formation and neocortical development.

Human molecular genetics, 29(2):248-263.

WDR62 mutations that result in protein loss, truncation or single amino-acid substitutions are causative for human microcephaly, indicating critical roles in cell expansion required for brain development. WDR62 missense mutations that retain protein expression represent partial loss-of-function mutants that may therefore provide specific insights into radial glial cell processes critical for brain growth. Here we utilized CRISPR/Cas9 approaches to generate three strains of WDR62 mutant mice; WDR62 V66M/V66M and WDR62R439H/R439H mice recapitulate conserved missense mutations found in humans with microcephaly, with the third strain being a null allele (WDR62stop/stop). Each of these mutations resulted in embryonic lethality to varying degrees and gross morphological defects consistent with ciliopathies (dwarfism, anophthalmia and microcephaly). We find that WDR62 mutant proteins (V66M and R439H) localize to the basal body but fail to recruit CPAP. As a consequence, we observe deficient recruitment of IFT88, a protein that is required for cilia formation. This underpins the maintenance of radial glia as WDR62 mutations caused premature differentiation of radial glia resulting in reduced generation of neurons and cortical thinning. These findings highlight the important role of the primary cilium in neocortical expansion and implicate ciliary dysfunction as underlying the pathology of MCPH2 patients.

RevDate: 2020-10-19
CmpDate: 2020-10-19

Tsai IC, Adams KA, Tzeng JA, et al (2019)

Genome-wide suppressor screen identifies USP35/USP38 as therapeutic candidates for ciliopathies.

JCI insight, 4(22):.

The ciliopathies are a group of phenotypically overlapping disorders caused by structural or functional defects in the primary cilium. Although disruption of numerous signaling pathways and cellular trafficking events have been implicated in ciliary pathology, treatment options for affected individuals remain limited. Here, we performed a genome-wide RNAi (RNA interference) screen to identify genetic suppressors of BBS4, one of the genes mutated in Bardet-Biedl syndrome (BBS). We discovered 10 genes that, when silenced, ameliorate BBS4-dependent pathology. One of these encodes USP35, a negative regulator of the ubiquitin proteasome system, suggesting that inhibition of a deubiquitinase, and subsequent facilitation of the clearance of signaling components, might ameliorate BBS-relevant phenotypes. Testing of this hypothesis in transient and stable zebrafish genetic models showed this posit to be true; suppression or ablation of usp35 ameliorated hallmark ciliopathy defects including impaired convergent extension (CE), renal tubule convolution, and retinal degeneration with concomitant clearance of effectors such as β-catenin and rhodopsin. Together, our findings reinforce a direct link between proteasome-dependent degradation and ciliopathies and suggest that augmentation of this system might offer a rational path to novel therapeutic modalities.

RevDate: 2020-10-20
CmpDate: 2020-10-20

Xiong Z, Xie Y, Yang Y, et al (2019)

Efficient gene correction of an aberrant splice site in β-thalassaemia iPSCs by CRISPR/Cas9 and single-strand oligodeoxynucleotides.

Journal of cellular and molecular medicine, 23(12):8046-8057.

β-thalassaemia is a prevalent hereditary haematological disease caused by mutations in the human haemoglobin β (HBB) gene. Among them, the HBB IVS2-654 (C > T) mutation, which is in the intron, creates an aberrant splicing site. Bone marrow transplantation for curing β-thalassaemia is limited due to the lack of matched donors. The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9), as a widely used tool for gene editing, is able to target specific sequence and create double-strand break (DSB), which can be combined with the single-stranded oligodeoxynucleotide (ssODN) to correct mutations. In this study, according to two different strategies, the HBB IVS2-654 mutation was seamlessly corrected in iPSCs by CRISPR/Cas9 system and ssODN. To reduce the occurrence of secondary cleavage, a more efficient strategy was adopted. The corrected iPSCs kept pluripotency and genome stability. Moreover, they could differentiate normally. Through CRISPR/Cas9 system and ssODN, our study provides improved strategies for gene correction of β-Thalassaemia, and the expression of the HBB gene can be restored, which can be used for gene therapy in the future.

RevDate: 2020-10-19
CmpDate: 2020-10-19

Vermersch E, Jouve C, JS Hulot (2020)

CRISPR/Cas9 gene-editing strategies in cardiovascular cells.

Cardiovascular research, 116(5):894-907.

Cardiovascular diseases are among the main causes of morbidity and mortality in Western countries and considered as a leading public health issue. Therefore, there is a strong need for new disease models to support the development of novel therapeutics approaches. The successive improvement of genome editing tools with zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and more recently with clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) has enabled the generation of genetically modified cells and organisms with much greater efficiency and precision than before. The simplicity of CRISPR/Cas9 technology made it especially suited for different studies, both in vitro and in vivo, and has been used in multiple studies evaluating gene functions, disease modelling, transcriptional regulation, and testing of novel therapeutic approaches. Notably, with the parallel development of human induced pluripotent stem cells (hiPSCs), the generation of knock-out and knock-in human cell lines significantly increased our understanding of mutation impacts and physiopathological mechanisms within the cardiovascular domain. Here, we review the recent development of CRISPR-Cas9 genome editing, the alternative tools, the available strategies to conduct genome editing in cardiovascular cells with a focus on its use for correcting mutations in vitro and in vivo both in germ and somatic cells. We will also highlight that, despite its potential, CRISPR/Cas9 technology comes with important technical and ethical limitations. The development of CRISPR/Cas9 genome editing for cardiovascular diseases indeed requires to develop a specific strategy in order to optimize the design of the genome editing tools, the manipulation of DNA repair mechanisms, the packaging and delivery of the tools to the studied organism, and the assessment of their efficiency and safety.

RevDate: 2020-10-19
CmpDate: 2020-10-19

Tran NT, Sommermann T, Graf R, et al (2019)

Efficient CRISPR/Cas9-Mediated Gene Knockin in Mouse Hematopoietic Stem and Progenitor Cells.

Cell reports, 28(13):3510-3522.e5.

Mutations accumulating in hematopoietic stem and progenitor cells (HSPCs) during development can cause severe hematological disorders. Modeling these mutations in mice is essential for understanding their functional consequences. Here, we describe an efficient CRISPR/Cas9-based system to knock in and repair genes in mouse HSPCs. CRISPR/Cas9 ribonucleoproteins, in combination with recombinant adeno-associated virus (rAAV)-DJ donor templates, led to gene knockin efficiencies of up to 30% in the Lmnb1 and Actb loci of mouse HSPCs in vitro. The targeted HSPCs engraft and reconstitute all immune cell lineages in the recipient mice. Using this approach, we corrected a neomycin-disrupted Rag2 gene. The Rag2-corrected HSPCs restore B and T cell development in vivo, confirming the functionality of the approach. Our method provides an efficient strategy to study gene function in the hematopoietic system and model hematological disorders in vivo, without the need for germline mutagenesis.

RevDate: 2020-10-20
CmpDate: 2020-10-20

Kumita W, Sato K, Suzuki Y, et al (2019)

Efficient generation of Knock-in/Knock-out marmoset embryo via CRISPR/Cas9 gene editing.

Scientific reports, 9(1):12719 pii:10.1038/s41598-019-49110-3.

Genetically modified nonhuman primates (NHP) are useful models for biomedical research. Gene editing technologies have enabled production of target-gene knock-out (KO) NHP models. Target-gene-KO/knock-in (KI) efficiency of CRISPR/Cas9 has not been extensively investigated in marmosets. In this study, optimum conditions for target gene modification efficacies of CRISPR/mRNA and CRISPR/nuclease in marmoset embryos were examined. CRISPR/nuclease was more effective than CRISPR/mRNA in avoiding mosaic genetic alteration. Furthermore, optimal conditions to generate KI marmoset embryos were investigated using CRISPR/Cas9 and 2 different lengths (36 nt and 100 nt) each of a sense or anti-sense single-strand oligonucleotide (ssODN). KIs were observed when CRISPR/nuclease and 36 nt sense or anti-sense ssODNs were injected into embryos. All embryos exhibited mosaic mutations with KI and KO, or imprecise KI, of c-kit. Although further improvement of KI strategies is required, these results indicated that CRISPR/Cas9 may be utilized to produce KO/KI marmosets via gene editing.

RevDate: 2020-10-19
CmpDate: 2020-10-19

Tsai SY, Ghazizadeh Z, Wang HJ, et al (2020)

A human embryonic stem cell reporter line for monitoring chemical-induced cardiotoxicity.

Cardiovascular research, 116(3):658-670.

AIMS: Human embryonic stem cells (hESCs) can be used to generate scalable numbers of cardiomyocytes (CMs) for studying cardiac biology, disease modelling, drug screens, and potentially for regenerative therapies. A fluorescence-based reporter line will significantly enhance our capacities to visualize the derivation, survival, and function of hESC-derived CMs. Our goal was to develop a reporter cell line for real-time monitoring of live hESC-derived CMs.

METHODS AND RESULTS: We used CRISPR/Cas9 to knock a mCherry reporter gene into the MYH6 locus of hESC lines, H1 and H9, enabling real-time monitoring of the generation of CMs. MYH6:mCherry+ cells express atrial or ventricular markers and display a range of cardiomyocyte action potential morphologies. At 20 days of differentiation, MYH6:mCherry+ cells show features characteristic of human CMs and can be used successfully to monitor drug-induced cardiotoxicity and oleic acid-induced cardiac arrhythmia.

CONCLUSION: We created two MYH6:mCherry hESC reporter lines and documented the application of these lines for disease modelling relevant to cardiomyocyte biology.

RevDate: 2020-10-20
CmpDate: 2020-10-20

Cecconi D, Brandi J, Manfredi M, et al (2019)

Runx2 stimulates neoangiogenesis through the Runt domain in melanoma.

Scientific reports, 9(1):8052 pii:10.1038/s41598-019-44552-1.

Runx2 is a transcription factor involved in melanoma cell migration and proliferation. Here, we extended the analysis of Runt domain of Runx2 in melanoma cells to deepen understanding of the underlying mechanisms. By the CRISPR/Cas9 system we generated the Runt KO melanoma cells 3G8. Interestingly, the proteome analysis showed a specific protein signature of 3G8 cells related to apoptosis and migration, and pointed out the involvement of Runt domain in the neoangiogenesis process. Among the proteins implicated in angiogenesis we identified fatty acid synthase, chloride intracellular channel protein-4, heat shock protein beta-1, Rho guanine nucleotide exchange factor 1, D-3-phosphoglycerate dehydrogenase, myosin-1c and caveolin-1. Upon querying the TCGA provisional database for melanoma, the genes related to these proteins were found altered in 51.36% of total patients. In addition, VEGF gene expression was reduced in 3G8 as compared to A375 cells; and HUVEC co-cultured with 3G8 cells expressed lower levels of CD105 and CD31 neoangiogenetic markers. Furthermore, the tube formation assay revealed down-regulation of capillary-like structures in HUVEC co-cultured with 3G8 in comparison to those with A375 cells. These findings provide new insight into Runx2 molecular details which can be crucial to possibly propose it as an oncotarget of melanoma.

RevDate: 2020-10-20
CmpDate: 2020-10-20

Watanabe M, Nakano K, Uchikura A, et al (2019)

Anephrogenic phenotype induced by SALL1 gene knockout in pigs.

Scientific reports, 9(1):8016 pii:10.1038/s41598-019-44387-w.

To combat organ shortage in transplantation medicine, a novel strategy has been proposed to generate human organs from exogenous pluripotent stem cells utilizing the developmental mechanisms of pig embryos/foetuses. Genetically modified pigs missing specific organs are key elements in this strategy. In this study, we demonstrate the feasibility of using a genome-editing approach to generate anephrogenic foetuses in a genetically engineered pig model. SALL1 knockout (KO) was successfully induced by injecting genome-editing molecules into the cytoplasm of pig zygotes, which generated the anephrogenic phenotype. Extinguished SALL1 expression and marked dysgenesis of nephron structures were observed in the rudimentary kidney tissue of SALL1-KO foetuses. Biallelic KO mutations of the target gene induced nephrogenic defects; however, biallelic mutations involving small in-frame deletions did not induce the anephrogenic phenotype. Through production of F1 progeny from mutant founder pigs, we identified mutations that could reliably induce the anephrogenic phenotype and hence established a line of fertile SALL1-mutant pigs. Our study lays important technical groundwork for the realization of human kidney regeneration through the use of an empty developmental niche in pig foetuses.

RevDate: 2020-10-19
CmpDate: 2020-10-19

Kataoka Y, Iimori M, Niimi S, et al (2019)

Cytotoxicity of trifluridine correlates with the thymidine kinase 1 expression level.

Scientific reports, 9(1):7964 pii:10.1038/s41598-019-44399-6.

Trifluridine (FTD), a tri-fluorinated thymidine analogue, is a key component of the oral antitumor drug FTD/TPI (also known as TAS-102), which is used to treat refractory metastatic colorectal cancer. Thymidine kinase 1 (TK1) is thought to be important for the incorporation of FTD into DNA, resulting in DNA dysfunction and cytotoxicity. However, it remains unknown whether TK1 is essential for FTD incorporation into DNA and whether this event is affected by the expression level of TK1 because TK1-specific-deficient human cancer cell lines have not been established. Here, we generated TK1-knock-out human colorectal cancer cells using the CRISPR/Cas9 genome editing system and validated the specificity of TK1 knock-out by measuring expression of AFMID, which is encoded on the same locus as TK1. Using TK1-knock-out cells, we confirmed that TK1 is essential for cellular sensitivity to FTD. Furthermore, we demonstrated a correlation between the TK1 expression level and cytotoxicity of FTD using cells with inducible TK1 expression, which were generated from TK1-knock-out cells. Based on our finding that the TK1 expression level correlates with sensitivity to FTD, we suggest that FTD/TPI might efficiently treat cancers with high TK1 expression.

RevDate: 2020-10-19
CmpDate: 2020-10-19

Huang Y, Ding Y, Liu Y, et al (2019)

Optimisation of the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 : single-guide RNA (sgRNA) delivery system in a goat model.

Reproduction, fertility, and development, 31(9):1533-1537.

The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system is an efficient method for the production of gene-edited animals. We have successfully generated gene-modified goats and sheep via zygote injection of Cas9 mRNA and single-guide RNA (sgRNA) mixtures. However, the delivery system for microinjection largely refers to methods established for mice; optimised injection conditions are urgently required for the generation of large animals. Here, we designed a study to optimise the Cas9 mRNA and sgRNA delivery system for goats. By comparing four computational tools for sgRNA design and validating the targeting efficiency in goat fibroblasts, we suggest a protocol for the selection of desirable sgRNAs with higher targeting efficiency and negligible off-target mutations. We further evaluated the editing efficiency in goat zygotes injected with Cas9:sgRNA (sg8) and found that injection with 50ngμL-1 Cas9 mRNA and 25ngμL-1 sgRNA yielded an increased editing efficiency. Our results provide a reference protocol for the optimisation of the injection conditions for the efficient editing of large animal genomes via the zygote injection approach.

RevDate: 2020-10-19

Zhang Z, Li Y, Luo L, et al (2020)

Corrigendum: Characterization of cmcp Gene as a Pathogenicity Factor of Ceratocystis manginecans.

Frontiers in microbiology, 11:595238.

[This corrects the article DOI: 10.3389/fmicb.2020.01824.].

RevDate: 2020-10-19

Yimer SA, Kalayou S, Homberset H, et al (2020)

Lineage-Specific Proteomic Signatures in the Mycobacterium tuberculosis Complex Reveal Differential Abundance of Proteins Involved in Virulence, DNA Repair, CRISPR-Cas, Bioenergetics and Lipid Metabolism.

Frontiers in microbiology, 11:550760.

Despite the discovery of the tubercle bacillus more than 130 years ago, its physiology and the mechanisms of virulence are still not fully understood. A comprehensive analysis of the proteomes of members of the human-adapted Mycobacterium tuberculosis complex (MTBC) lineages 3, 4, 5, and 7 was conducted to better understand the evolution of virulence and other physiological characteristics. Unique and shared proteomic signatures in these modern, pre-modern and ancient MTBC lineages, as deduced from quantitative bioinformatics analyses of high-resolution mass spectrometry data, were delineated. The main proteomic findings were verified by using immunoblotting. In addition, analysis of multiple genome alignment of members of the same lineages was performed. Label-free peptide quantification of whole cells from MTBC lineages 3, 4, 5, and 7 yielded a total of 38,346 unique peptides derived from 3092 proteins, representing 77% coverage of the predicted proteome. MTBC lineage-specific differential expression was observed for 539 proteins. Lineage 7 exhibited a markedly reduced abundance of proteins involved in DNA repair, type VII ESX-3 and ESX-1 secretion systems, lipid metabolism and inorganic phosphate uptake, and an increased abundance of proteins involved in alternative pathways of the TCA cycle and the CRISPR-Cas system as compared to the other lineages. Lineages 3 and 4 exhibited a higher abundance of proteins involved in virulence, DNA repair, drug resistance and other metabolic pathways. The high throughput analysis of the MTBC proteome by super-resolution mass spectrometry provided an insight into the differential expression of proteins between MTBC lineages 3, 4, 5, and 7 that may explain the slow growth and reduced virulence, metabolic flexibility, and the ability to survive under adverse growth conditions of lineage 7.

RevDate: 2020-10-17

Huang L, Yang B, Yi H, et al (2020)

AcrDB: a database of anti-CRISPR operons in prokaryotes and viruses.

Nucleic acids research pii:5929236 [Epub ahead of print].

CRISPR-Cas is an anti-viral mechanism of prokaryotes that has been widely adopted for genome editing. To make CRISPR-Cas genome editing more controllable and safer to use, anti-CRISPR proteins have been recently exploited to prevent excessive/prolonged Cas nuclease cleavage. Anti-CRISPR (Acr) proteins are encoded by (pro)phages/(pro)viruses, and have the ability to inhibit their host's CRISPR-Cas systems. We have built an online database AcrDB (http://bcb.unl.edu/AcrDB) by scanning ∼19 000 genomes of prokaryotes and viruses with AcrFinder, a recently developed Acr-Aca (Acr-associated regulator) operon prediction program. Proteins in Acr-Aca operons were further processed by two machine learning-based programs (AcRanker and PaCRISPR) to obtain numerical scores/ranks. Compared to other anti-CRISPR databases, AcrDB has the following unique features: (i) It is a genome-scale database with the largest collection of data (39 799 Acr-Aca operons containing Aca or Acr homologs); (ii) It offers a user-friendly web interface with various functions for browsing, graphically viewing, searching, and batch downloading Acr-Aca operons; (iii) It focuses on the genomic context of Acr and Aca candidates instead of individual Acr protein family and (iv) It collects data with three independent programs each having a unique data mining algorithm for cross validation. AcrDB will be a valuable resource to the anti-CRISPR research community.

RevDate: 2020-10-17

Chan AN, Wang LL, Zhu YJ, et al (2020)

Identification through fine mapping and verification using CRISPR/Cas9-targeted mutagenesis for a minor QTL controlling grain weight in rice.

TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik pii:10.1007/s00122-020-03699-6 [Epub ahead of print].

KEY MESSAGE: A minor QTL for grain weight in rice, qTGW1.2b, was fine-mapped. Its casual gene OsVQ4 was confirmed through CRISPR/Cas9-targeted mutagenesis, exhibiting an effect that was larger than the original QTL effect. The CRISPR/Cas system exhibits a great potential for rice improvement, but the application was severely hindered due to insufficient target genes, especial the lack of validated genes underlying quantitative trait loci having small effects. In this study, a minor QTL for grain weight, qTGW1.2b, was fine-mapped into a 44.0 kb region using seven sets of near isogenic lines (NILs) developed from the indica rice cross (Zhenshan 97)3/Milyang 46, followed by validation of the causal gene using CRISPR/Cas9-targeted mutagenesis. In the NIL populations, 1000-grain weight of the Zhenshan 97 homozygous lines decreased by 0.9-2.0% compared with the Milyang 46 homozygous lines. A gene encoding VQ-motif protein, OsVQ4, was identified as the candidate gene based on parental sequence differences. The effect of OsVQ4 was confirmed by creating CRISPR/Cas9 knockout lines, whose 1000-grain weight decreased by 2.8-9.8% compared with the wild-type transgenic line and the recipient. These results indicate that applying genome editing system could create novel alleles with large phenotypic variation at minor QTLs, which is an effective way to validate causal genes of minor QTLs. Our study establishes a strategy for cloning minor QTLs, which could also be used to identify a large number of potential target genes for the application of CRISPR/Cas system.

RevDate: 2020-10-19

Huang X, Sun W, Cheng Z, et al (2020)

Structural basis for two metal-ion catalysis of DNA cleavage by Cas12i2.

Nature communications, 11(1):5241 pii:10.1038/s41467-020-19072-6.

To understand how the RuvC catalytic domain of Class 2 Cas proteins cleaves DNA, it will be necessary to elucidate the structures of RuvC-containing Cas complexes in their catalytically competent states. Cas12i2 is a Class 2 type V-I CRISPR-Cas endonuclease that cleaves target dsDNA by an unknown mechanism. Here, we report structures of Cas12i2-crRNA-DNA complexes and a Cas12i2-crRNA complex. We reveal the mechanism of DNA recognition and cleavage by Cas12i2, and activation of the RuvC catalytic pocket induced by a conformational change of the Helical-II domain. The seed region (nucleotides 1-8) is dispensable for RuvC activation, but the duplex of the central spacer (nucleotides 9-15) is required. We captured the catalytic state of Cas12i2, with both metal ions and the ssDNA substrate bound in the RuvC catalytic pocket. Together, our studies provide significant insights into the DNA cleavage mechanism by RuvC-containing Cas proteins.

RevDate: 2020-10-19

Saha C, Horst-Kreft D, Kross I, et al (2020)

Campylobacter jejuni Cas9 Modulates the Transcriptome in Caco-2 Intestinal Epithelial Cells.

Genes, 11(10): pii:genes11101193.

The zoonotic human pathogen Campylobacter jejuni is known for its ability to induce DNA-damage and cell death pathology in humans. The molecular mechanism behind this phenomenon involves nuclear translocation by Cas9, a nuclease in C. jejuni (CjeCas9) that is the molecular marker of the Type II CRISPR-Cas system. However, it is unknown via which cellular pathways CjeCas9 drives human intestinal epithelial cells into cell death. Here, we show that CjeCas9 released by C. jejuni during the infection of Caco-2 human intestinal epithelial cells directly modulates Caco-2 transcriptomes during the first four hours of infection. Specifically, our results reveal that CjeCas9 activates DNA damage (p53, ATM (Ataxia Telangiectasia Mutated Protein)), pro-inflammatory (NF-κB (Nuclear factor-κB)) signaling and cell death pathways, driving Caco-2 cells infected by wild-type C. jejuni, but not when infected by a cas9 deletion mutant, towards programmed cell death. This work corroborates our previous finding that CjeCas9 is cytotoxic and highlights on a RNA level the basal cellular pathways that are modulated.

RevDate: 2020-10-15

Kondrateva E, Demchenko A, Lavrov A, et al (2020)

An overview of currently available molecular Cas-tools for precise genome modification.

Gene pii:S0378-1119(20)30894-5 [Epub ahead of print].

CRISPR-Cas system was first mentioned in 1987, and over the years have been studied so active that now it becomes the state-of-theart tool for genome editing. Its working principle is based on Cas nuclease ability to bind short RNA, which targets it to complementary DNA or RNA sequence for highly precise cleavage. This alone or together with donor DNA allows to modify targeted sequence in different ways. Considering the many limitations of using native CRISPR-Cas systems, scientists around the world are working on creating modified variants to improve their specificity and efficiency in different objects. In addition, the use of the Cas effectors' targeting function in complex systems with other proteins is a promising work direction, as a result of which new tools are created with features such as single base editing, editing DNA without break and donor DNA, activation and repression of transcription, epigenetic regulation, modifying of different repair pathways involvement etc. In this review, we decided to consider in detail exactly this issue of variants of Cas effectors, their modifications and fusion molecules, which improve DNA-targeting and expand the scope of Cas effectors.

RevDate: 2020-10-16
CmpDate: 2020-10-16

Lundin A, Porritt MJ, Jaiswal H, et al (2020)

Development of an ObLiGaRe Doxycycline Inducible Cas9 system for pre-clinical cancer drug discovery.

Nature communications, 11(1):4903 pii:10.1038/s41467-020-18548-9.

The CRISPR-Cas9 system has increased the speed and precision of genetic editing in cells and animals. However, model generation for drug development is still expensive and time-consuming, demanding more target flexibility and faster turnaround times with high reproducibility. The generation of a tightly controlled ObLiGaRe doxycycline inducible SpCas9 (ODInCas9) transgene and its use in targeted ObLiGaRe results in functional integration into both human and mouse cells culminating in the generation of the ODInCas9 mouse. Genomic editing can be performed in cells of various tissue origins without any detectable gene editing in the absence of doxycycline. Somatic in vivo editing can model non-small cell lung cancer (NSCLC) adenocarcinomas, enabling treatment studies to validate the efficacy of candidate drugs. The ODInCas9 mouse allows robust and tunable genome editing granting flexibility, speed and uniformity at less cost, leading to high throughput and practical preclinical in vivo therapeutic testing.

RevDate: 2020-10-16
CmpDate: 2020-10-16

He X, Liu C, Yang X, et al (2020)

Bi-allelic Loss-of-function Variants in CFAP58 Cause Flagellar Axoneme and Mitochondrial Sheath Defects and Asthenoteratozoospermia in Humans and Mice.

American journal of human genetics, 107(3):514-526.

Multiple morphological abnormalities of the sperm flagella (MMAF) is a severe form of asthenoteratozoospermia. Although recent studies have revealed several MMAF-associated genes and demonstrated MMAF to be a genetically heterogeneous disease, at least one-third of the cases are still not well understood for their etiology. Here, we identified bi-allelic loss-of-function variants in CFAP58 by using whole-exome sequencing in five (5.6%) unrelated individuals from a cohort of 90 MMAF-affected Chinese men. Each of the men harboring bi-allelic CFAP58 variants presented typical MMAF phenotypes. Transmission electron microscopy demonstrated striking flagellar defects with axonemal and mitochondrial sheath malformations. CFAP58 is predominantly expressed in the testis and encodes a cilia- and flagella-associated protein. Immunofluorescence assays showed that CFAP58 localized at the entire flagella of control sperm and predominantly concentrated in the mid-piece. Immunoblotting and immunofluorescence assays showed that the abundances of axoneme ultrastructure markers SPAG6 and SPEF2 and a mitochondrial sheath protein, HSP60, were significantly reduced in the spermatozoa from men harboring bi-allelic CFAP58 variants. We generated Cfap58-knockout mice via CRISPR/Cas9 technology. The male mice were infertile and presented with severe flagellar defects, consistent with the sperm phenotypes in MMAF-affected men. Overall, our findings in humans and mice strongly suggest that CFAP58 plays a vital role in sperm flagellogenesis and demonstrate that bi-allelic loss-of-function variants in CFAP58 can cause axoneme and peri-axoneme malformations leading to male infertility. This study provides crucial insights for understanding and counseling of MMAF-associated asthenoteratozoospermia.

RevDate: 2020-10-15
CmpDate: 2020-10-15

Karimian A, Gorjizadeh N, Alemi F, et al (2020)

CRISPR/Cas9 novel therapeutic road for the treatment of neurodegenerative diseases.

Life sciences, 259:118165.

CRISPR (clustered regularly interspaced short palindromic Repeats)/Cas9 is a new genetic editing technology that can be a beneficial method to advance gene therapy. CRISPR technology is a defense system of some bacteria against invading viruses. Genome editing based on the CRISPR/Cas9 system is an efficient and potential technology that can be a viable alternative to traditional methods. This system is a compound of a short guide RNAs (gRNAs) for identifying the target DNA sequence and Cas9 protein as nuclease for breaking and cutting of DNA. In this review, recent advances in the CRISPR/Cas9-mediated genome editing tools are presented as well as their use in gene therapy strategies for the treatment of neurological disorders including Parkinson's disease, Alzheimer's disease, and Huntington's disease.

RevDate: 2020-10-15
CmpDate: 2020-10-15

Kim H, Lee WJ, Oh Y, et al (2020)

Enhancement of target specificity of CRISPR-Cas12a by using a chimeric DNA-RNA guide.

Nucleic acids research, 48(15):8601-8616.

The CRISPR-Cas9 system is widely used for target-specific genome engineering. CRISPR-Cas12a (Cpf1) is one of the CRISPR effectors that controls target genes by recognizing thymine-rich protospacer adjacent motif (PAM) sequences. Cas12a has a higher sensitivity to mismatches in the guide RNA than does Cas9; therefore, off-target sequence recognition and cleavage are lower. However, it tolerates mismatches in regions distant from the PAM sequence (TTTN or TTN) in the protospacer, and off-target cleavage issues may become more problematic when Cas12a activity is improved for therapeutic purposes. Therefore, we investigated off-target cleavage by Cas12a and modified the Cas12a (cr)RNA to address the off-target cleavage issue. We developed a CRISPR-Cas12a that can induce mutations in target DNA sequences in a highly specific and effective manner by partially substituting the (cr)RNA with DNA to change the energy potential of base pairing to the target DNA. A model to explain how chimeric (cr)RNA guided CRISPR-Cas12a and SpCas9 nickase effectively work in the intracellular genome is suggested. Chimeric guide-based CRISPR- Cas12a genome editing with reduced off-target cleavage, and the resultant, increased safety has potential for therapeutic applications in incurable diseases caused by genetic mutations.

RevDate: 2020-10-14
CmpDate: 2020-10-14

Choi J, Diao H, Faliti CE, et al (2020)

Bcl-6 is the nexus transcription factor of T follicular helper cells via repressor-of-repressor circuits.

Nature immunology, 21(7):777-789.

T follicular helper (TFH) cells are a distinct type of CD4+ T cells that are essential for most antibody and B lymphocyte responses. TFH cell regulation and dysregulation is involved in a range of diseases. Bcl-6 is the lineage-defining transcription factor of TFH cells and its activity is essential for TFH cell differentiation and function. However, how Bcl-6 controls TFH biology has largely remained unclear, at least in part due to the intrinsic challenges of connecting repressors to gene upregulation in complex cell types with multiple possible differentiation fates. Multiple competing models were tested here by a series of experimental approaches to determine that Bcl-6 exhibits negative autoregulation and controls pleiotropic attributes of TFH differentiation and function, including migration, costimulation, inhibitory receptors and cytokines, via multiple repressor-of-repressor gene circuits.

RevDate: 2020-10-15
CmpDate: 2020-10-15

Sharma S, GJ Wright (2020)

Cell Surface Receptor Identification Using Genome-Scale CRISPR/Cas9 Genetic Screens.

Journal of visualized experiments : JoVE.

Intercellular communication mediated by direct interactions between membrane-embedded cell surface receptors is crucial for the normal development and functioning of multicellular organisms. Detecting these interactions remains technically challenging, however. This manuscript describes a systematic genome-scale CRISPR/Cas9 knockout genetic screening approach that reveals cellular pathways required for specific cell surface recognition events. This assay utilizes recombinant proteins produced in a mammalian protein expression system as avid binding probes to identify interaction partners in a cell-based genetic screen. This method can be used to identify the genes necessary for cell surface interactions detected by recombinant binding probes corresponding to the ectodomains of membrane-embedded receptors. Importantly, given the genome-scale nature of this approach, it also has the advantage of not only identifying the direct receptor but also the cellular components that are required for the presentation of the receptor at the cell surface, thereby providing valuable insights into the biology of the receptor.

RevDate: 2020-10-14
CmpDate: 2020-10-14

Masuda T, Amann L, Sankowski R, et al (2020)

Novel Hexb-based tools for studying microglia in the CNS.

Nature immunology, 21(7):802-815.

Microglia and central nervous system (CNS)-associated macrophages (CAMs), such as perivascular and meningeal macrophages, are implicated in virtually all diseases of the CNS. However, little is known about their cell-type-specific roles in the absence of suitable tools that would allow for functional discrimination between the ontogenetically closely related microglia and CAMs. To develop a new microglia gene targeting model, we first applied massively parallel single-cell analyses to compare microglia and CAM signatures during homeostasis and disease and identified hexosaminidase subunit beta (Hexb) as a stably expressed microglia core gene, whereas other microglia core genes were substantially downregulated during pathologies. Next, we generated HexbtdTomato mice to stably monitor microglia behavior in vivo. Finally, the Hexb locus was employed for tamoxifen-inducible Cre-mediated gene manipulation in microglia and for fate mapping of microglia but not CAMs. In sum, we provide valuable new genetic tools to specifically study microglia functions in the CNS.

RevDate: 2020-10-16
CmpDate: 2020-10-16

Todokoro T, Bando H, Kotaka A, et al (2020)

Identification of a novel pyrithiamine resistance marker gene thiI for genome co-editing in Aspergillus oryzae.

Journal of bioscience and bioengineering, 130(3):227-232.

Marker genes are essential for gene modification and genome editing of microorganisms. In Aspergillus oryzae, a widely used host for enzyme production, only a few marker genes can be used for positive selection. One of these genes, the pyrithiamine (PT) resistance marker gene thiA, is not useful for CRISPR/Cas9 genome editing because of its unique resistance-conferring mechanism. In this study, a novel PT resistance marker was investigated considering its potential applications in genome editing. A mutant resistant to PT was selected from UV-mutagenized A. oryzae RIB40. Whole genome analysis was conducted on the mutants, and a novel candidate gene for PT resistance was identified. This candidate gene exhibited similarity to the thiamine transporter gene thi9 of Schizosaccharomyces pombe and was designated as thiI. A thiI loss-of-function mutant was generated using the CRISPR/Cas9 genome editing system to investigate its effect on PT resistance. This mutant showed PT resistance and exhibited no growth defect or auxotrophy. The thiI gene was further investigated for its use as a selection marker in genome co-editing. Ribonucleoprotein complex comprising recombinant Cas9 nuclease and sgRNA targeting thiI or another target gene (wA or sreA) was prepared and simultaneously introduced into A. oryzae RIB40. thiI and target gene double loss-of-function mutants were efficiently selected on PT-containing medium. thiI was shown to be a useful marker gene in A. oryzae for use in genome editing. This study is expected to provide insights, which will promote basic research and industrial applications of A. oryzae.

RevDate: 2020-10-15
CmpDate: 2020-10-15

Hampton T (2020)

With First CRISPR Trials, Gene Editing Moves Toward the Clinic.

JAMA, 323(16):1537-1539.

RevDate: 2020-10-16
CmpDate: 2020-10-16

Wang D, Zhang F, G Gao (2020)

CRISPR-Based Therapeutic Genome Editing: Strategies and In Vivo Delivery by AAV Vectors.

Cell, 181(1):136-150.

The development of clustered regularly interspaced short-palindromic repeat (CRISPR)-based biotechnologies has revolutionized the life sciences and introduced new therapeutic modalities with the potential to treat a wide range of diseases. Here, we describe CRISPR-based strategies to improve human health, with an emphasis on the delivery of CRISPR therapeutics directly into the human body using adeno-associated virus (AAV) vectors. We also discuss challenges facing broad deployment of CRISPR-based therapeutics and highlight areas where continued discovery and technological development can further advance these revolutionary new treatments.

RevDate: 2020-10-16
CmpDate: 2020-10-16

Liu Y, Xu Z, Zhang Y, et al (2020)

Marek's disease virus as a CRISPR/Cas9 delivery system to defend against avian leukosis virus infection in chickens.

Veterinary microbiology, 242:108589.

The CRISPR/CRISPR-associated protein 9 (Cas9) system is a powerful gene-editing tool originally discovered as an integral mediator of bacterial adaptive immunity. Recently, this technology has been explored for its potential utility in providing new and unique treatments for viral infection. Marek's disease virus (MDV) and avian leukosis virus subgroup J (ALV-J), major immunosuppressive viruses, cause significant economic losses to the chicken industry. Here, we evaluated the efficacy of using MDV as a CRISPR/Cas9-delivery system to directly target and disrupt the reverse-transcribed products of the ALV-J RNA genome during its infection cycle in vitro and in vivo. We first screened multiple potential guide RNA (gRNA) target sites in the ALV-J genome and identified several optimized targets capable of effectively disrupting the latently integrated viral genome and providing efficient defense against new infection by ALV-J in cells. The optimal single-gRNAs and Cas9-expression cassettes were inserted into the genome of an MDV vaccine strain. The results indicated that engineered MDV stably expressing ALV-J-targeting CRISPR/Cas9 efficiently resisted ALV-J challenge in host cells. These findings demonstrated the CRISPR/Cas9 system as an effective treatment strategy against ALV-J infection. Furthermore, the results highlighted the potential of MDV as an effective delivery system for CRISPR/Cas9 in chickens.

RevDate: 2020-10-14
CmpDate: 2020-10-14

Kurihara T, Kouyama-Suzuki E, Satoga M, et al (2020)

DNA repair protein RAD51 enhances the CRISPR/Cas9-mediated knock-in efficiency in brain neurons.

Biochemical and biophysical research communications, 524(3):621-628.

Gene knock-in using the CRISPR/Cas9 system can be achieved in a specific population of neurons in the mouse brain, by using in utero electroporation to introduce DNA fragments into neural progenitor cells. Using this strategy, we previously knocked-in the EGFP coding sequence into the N-terminal region of the β-actin gene specifically in the pyramidal neurons in layer 2/3 of the somatosensory cortex. However, the knock-in efficiency was less than 2% of the transfected neurons. In this study, we sought to improve the knock-in efficiency using this system. First, we varied the length of the homology arms of the β-actin donor template DNA, and found that the knock-in efficiency was increased to ∼14% by extending the length of the 5' and 3' homology arms to 1.6 kb and 2.0 kb, respectively. We then tested the effect of the DNA repair protein RAD51 and the knock-in efficiency was increased up to 2.5-fold when co-transfecting with two different β-actin and a camk2a targeting EGFP knock-in modules. The RAD51 overexpression did not alter the migration of developing neurons, density or morphology of the dendritic spines compared to those in neurons not transfected with RAD51. RAD51 expression will be useful for increasing the knock-in efficiency in neurons in vivo by CRISPR/Cas9-mediated homology directed repair (HDR).

RevDate: 2020-10-14
CmpDate: 2020-10-14

Moreira D, Pereira AM, Lopes AL, et al (2020)

The best CRISPR/Cas9 versus RNA interference approaches for Arabinogalactan proteins' study.

Molecular biology reports, 47(3):2315-2325.

Arabinogalactan Proteins (AGPs) are hydroxyproline-rich proteins containing a high proportion of carbohydrates, widely spread in the plant kingdom. AGPs have been suggested to play important roles in plant development processes, especially in sexual plant reproduction. Nevertheless, the functions of a large number of these molecules, remains to be discovered. In this review, we discuss two revolutionary genetic techniques that are able to decode the roles of these glycoproteins in an easy and efficient way. The RNA interference is a frequently technique used in plant biology that promotes genes silencing. The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated protein 9 (CRISPR/Cas9), emerged a few years ago as a revolutionary genome-editing technique that has allowed null mutants to be obtained in a wide variety of organisms, including plants. The two techniques have some differences between them and depending on the research objective, these may work as advantage or disadvantage. In the present work, we propose the use of the two techniques to obtain AGP mutants easily and quickly, helping to unravel the role of AGPs, surely a great asset for the future.

RevDate: 2020-10-14
CmpDate: 2020-10-14

Tsapara G, Andermatt I, ET Stoeckli (2020)

Gene Silencing in Chicken Brain Development.

Methods in molecular biology (Clifton, N.J.), 2047:439-456.

Despite the development of brain organoids and neural cultures derived from iPSCs (induced pluripotent stem cells), brain development can only be studied in an animal. The mouse is the most commonly used vertebrate model for the analysis of gene function because of the well-established genetic tools that are available for loss-of-function studies. However, studies of gene function during development can be problematic in mammals. Many genes are active during different stages of development. Absence of gene function during early development may cause aberrant neurogenesis or even embryonic lethality and thus prevent analysis of later stages of development. To avoid these problems, precise temporal control of gene silencing is required.In contrast to mammals, oviparous animals are accessible for experimental manipulations during embryonic development. The combination of accessibility and RNAi- or Crispr/Cas9-based gene silencing makes the chicken embryo a powerful model for developmental studies. Depending on the time window during which gene silencing is attempted, chicken embryos can be used in ovo or ex ovo in a domed dish for easier access during later stages of development. Both techniques allow for precise temporal control of gene silencing during embryonic development.

RevDate: 2020-10-15
CmpDate: 2020-10-15

Bocharnikov AV, Keegan J, Wacleche VS, et al (2019)

PD-1hiCXCR5- T peripheral helper cells promote B cell responses in lupus via MAF and IL-21.

JCI insight, 4(20):.

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by pathologic T cell-B cell interactions and autoantibody production. Defining the T cell populations that drive B cell responses in SLE may enable design of therapies that specifically target pathologic cell subsets. Here, we evaluated the phenotypes of CD4+ T cells in the circulation of 52 SLE patients drawn from multiple cohorts and identified a highly expanded PD-1hiCXCR5-CD4+ T cell population. Cytometric, transcriptomic, and functional assays demonstrated that PD-1hiCXCR5-CD4+ T cells from SLE patients are T peripheral helper (Tph) cells, a CXCR5- T cell population that stimulates B cell responses via IL-21. The frequency of Tph cells, but not T follicular helper (Tfh) cells, correlated with both clinical disease activity and the frequency of CD11c+ B cells in SLE patients. PD-1hiCD4+ T cells were found within lupus nephritis kidneys and correlated with B cell numbers in the kidney. Both IL-21 neutralization and CRISPR-mediated deletion of MAF abrogated the ability of Tph cells to induce memory B cell differentiation into plasmablasts in vitro. These findings identify Tph cells as a highly expanded T cell population in SLE and suggest a key role for Tph cells in stimulating pathologic B cell responses.

RevDate: 2020-10-15
CmpDate: 2020-10-15

Rovira Gonzalez YI, Moyer AL, LeTexier NJ, et al (2019)

Mss51 deletion enhances muscle metabolism and glucose homeostasis in mice.

JCI insight, 4(20):.

Myostatin is a negative regulator of muscle growth and metabolism and its inhibition in mice improves insulin sensitivity, increases glucose uptake into skeletal muscle, and decreases total body fat. A recently described mammalian protein called MSS51 is significantly downregulated with myostatin inhibition. In vitro disruption of Mss51 results in increased levels of ATP, β-oxidation, glycolysis, and oxidative phosphorylation. To determine the in vivo biological function of Mss51 in mice, we disrupted the Mss51 gene by CRISPR/Cas9 and found that Mss51-KO mice have normal muscle weights and fiber-type distribution but reduced fat pads. Myofibers isolated from Mss51-KO mice showed an increased oxygen consumption rate compared with WT controls, indicating an accelerated rate of skeletal muscle metabolism. The expression of genes related to oxidative phosphorylation and fatty acid β-oxidation were enhanced in skeletal muscle of Mss51-KO mice compared with that of WT mice. We found that mice lacking Mss51 and challenged with a high-fat diet were resistant to diet-induced weight gain, had increased whole-body glucose turnover and glycolysis rate, and increased systemic insulin sensitivity and fatty acid β-oxidation. These findings demonstrate that MSS51 modulates skeletal muscle mitochondrial respiration and regulates whole-body glucose and fatty acid metabolism, making it a potential target for obesity and diabetes.

RevDate: 2020-10-15
CmpDate: 2020-10-15

Merola J, Reschke M, Pierce RW, et al (2019)

Progenitor-derived human endothelial cells evade alloimmunity by CRISPR/Cas9-mediated complete ablation of MHC expression.

JCI insight, 4(20):.

Tissue engineering may address organ shortages currently limiting clinical transplantation. Off-the-shelf engineered vascularized organs will likely use allogeneic endothelial cells (ECs) to construct microvessels required for graft perfusion. Vasculogenic ECs can be differentiated from committed progenitors (human endothelial colony-forming cells or HECFCs) without risk of mutation or teratoma formation associated with reprogrammed stem cells. Like other ECs, these cells can express both class I and class II major histocompatibility complex (MHC) molecules, bind donor-specific antibody (DSA), activate alloreactive T effector memory cells, and initiate rejection in the absence of donor leukocytes. CRISPR/Cas9-mediated dual ablation of β2-microglobulin and class II transactivator (CIITA) in HECFC-derived ECs eliminates both class I and II MHC expression while retaining EC functions and vasculogenic potential. Importantly, dually ablated ECs no longer bind human DSA or activate allogeneic CD4+ effector memory T cells and are resistant to killing by CD8+ alloreactive cytotoxic T lymphocytes in vitro and in vivo. Despite absent class I MHC molecules, these ECs do not activate or elicit cytotoxic activity from allogeneic natural killer cells. These data suggest that HECFC-derived ECs lacking MHC molecule expression can be utilized for engineering vascularized grafts that evade allorejection.

RevDate: 2020-10-15
CmpDate: 2020-10-15

Makokha GN, Abe-Chayama H, Chowdhury S, et al (2019)

Regulation of the Hepatitis B virus replication and gene expression by the multi-functional protein TARDBP.

Scientific reports, 9(1):8462 pii:10.1038/s41598-019-44934-5.

Hepatitis B virus (HBV) infects the liver and is a key risk factor for hepatocellular carcinoma. Identification of host factors that support viral replication is important to understand mechanisms of viral replication and to develop new therapeutic strategies. We identified TARDBP as a host factor that regulates HBV. Silencing or knocking out the protein in HBV infected cells severely impaired the production of viral replicative intermediates, mRNAs, proteins, and virions, whereas ectopic expression of TARDBP rescued production of these products. Mechanistically, we found that the protein binds to the HBV core promoter, as shown by chromatin precipitation as well as mutagenesis and protein-DNA interaction assays. Using LC-MS/MS analysis, we also found that TARDBP binds to a number of other proteins known to support the HBV life cycle, including NPM1, PARP1, Hsp90, HNRNPC, SFPQ, PTBP1, HNRNPK, and PUF60. Interestingly, given its key role as a regulator of RNA splicing, we found that TARDBP has an inhibitory role on pregenomic RNA splicing, which might help the virus to export its non-canonical RNAs from the nucleus without being subjected to unwanted splicing, even though mRNA nuclear export is normally closely tied to RNA splicing. Taken together, our results demonstrate that TARDBP is involved in multiple steps of HBV replication via binding to both HBV DNA and RNA. The protein's broad interactome suggests that TARDBP may function as part of a RNA-binding scaffold involved in HBV replication and that the interaction between these proteins might be a target for development of anti-HBV drugs.

RevDate: 2020-10-16
CmpDate: 2020-10-16

Tamura M, Yonezawa T, Liu X, et al (2019)

Opposing effects of acute versus chronic inhibition of p53 on decitabine's efficacy in myeloid neoplasms.

Scientific reports, 9(1):8171 pii:10.1038/s41598-019-44496-6.

Decitabine is a DNA methyltransferase inhibitor and is considered a promising drug to treat myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML) with p53 mutations. However, whether loss of p53 in fact increases the response of MDS/AML cells to decitabine remains unclear. In this study, we assessed the role of p53 in MDS and AML cells treated with decitabine using mouse models for MLL-AF9-driven AML and mutant ASXL1-driven MDS/AML. CRISPR/Cas9-mediated depletion of p53 in MDS/AML cells did not increase, but rather decreased their sensitivity to decitabine. Forced expression of a dominant-negative p53 fragment (p53DD) in these cells also decreased their responses to decitabine, confirming that acute inhibition of p53 conferred resistance to decitabine in AML and MDS/AML cells. In contrast, MLL-AF9-expressing AML cells generated from bone marrow progenitors of Trp53-deficient mice were more sensitive to decitabine in vivo than their wild-type counterparts, suggesting that long-term chronic p53 deficiency increases decitabine sensitivity in AML cells. Taken together, these data revealed a multifaceted role for p53 to regulate responses of myeloid neoplasms to decitabine treatment.

RevDate: 2020-10-14
CmpDate: 2020-10-14

Sarr A, Bré J, Um IH, et al (2019)

Genome-scale CRISPR/Cas9 screen determines factors modulating sensitivity to ProTide NUC-1031.

Scientific reports, 9(1):7643 pii:10.1038/s41598-019-44089-3.

Gemcitabine is a fluoropyrimidine analogue that is used as a mainstay of chemotherapy treatment for pancreatic and ovarian cancers, amongst others. Despite its widespread use, gemcitabine achieves responses in less than 10% of patients with metastatic pancreatic cancer and has a very limited impact on overall survival due to intrinsic and acquired resistance. NUC-1031 (Acelarin), a phosphoramidate transformation of gemcitabine, was the first anti-cancer ProTide to enter the clinic. We find it displays important in vitro cytotoxicity differences to gemcitabine, and a genome-wide CRISPR/Cas9 genetic screening approach identified only the pyrimidine metabolism pathway as modifying cancer cell sensitivity to NUC-1031. Low deoxycytidine kinase expression in tumour biopsies from patients treated with gemcitabine, assessed by immunostaining and image analysis, correlates with a poor prognosis, but there is no such correlation in tumour biopsies from a Phase I cohort treated with NUC-1031.

RevDate: 2020-10-14
CmpDate: 2020-10-14

Stanic K, Reig G, Figueroa RJ, et al (2019)

The Reprimo gene family member, reprimo-like (rprml), is required for blood development in embryonic zebrafish.

Scientific reports, 9(1):7131 pii:10.1038/s41598-019-43436-8.

The Reprimo gene family comprises a group of single-exon genes for which their physiological function remains poorly understood. Heretofore, mammalian Reprimo (RPRM) has been described as a putative p53-dependent tumor suppressor gene that functions at the G2/M cell cycle checkpoint. Another family member, Reprimo-like (RPRML), has not yet an established role in physiology or pathology. Importantly, RPRML expression pattern is conserved between zebrafish and human species. Here, using CRISPR-Cas9 and antisense morpholino oligonucleotides, we disrupt the expression of rprml in zebrafish and demonstrate that its loss leads to impaired definitive hematopoiesis. The formation of hemangioblasts and the primitive wave of hematopoiesis occur normally in absence of rprml. Later in development there is a significant reduction in erythroid-myeloid precursors (EMP) at the posterior blood island (PBI) and a significant decline of definitive hematopoietic stem/progenitor cells (HSPCs). Furthermore, loss of rprml also increases the activity of caspase-3 in endothelial cells within the caudal hematopoietic tissue (CHT), the first perivascular niche where HSPCs reside during zebrafish embryonic development. Herein, we report an essential role for rprml during hematovascular development in zebrafish embryos, specifically during the definitive waves of hematopoiesis, indicating for the first time a physiological role for the rprml gene.

RevDate: 2020-10-16
CmpDate: 2020-10-16

Calatayud C, Carola G, Fernández-Carasa I, et al (2019)

CRISPR/Cas9-mediated generation of a tyrosine hydroxylase reporter iPSC line for live imaging and isolation of dopaminergic neurons.

Scientific reports, 9(1):6811 pii:10.1038/s41598-019-43080-2.

Patient-specific induced pluripotent stem cells (iPSCs) are a powerful tool to investigate the molecular mechanisms underlying Parkinson's disease (PD), and might provide novel platforms for systematic drug screening. Several strategies have been developed to generate iPSC-derived tyrosine hydroxylase (TH)-positive dopaminergic neurons (DAn), the clinically relevant cell type in PD; however, they often result in mixed neuronal cultures containing only a small proportion of TH-positive DAn. To overcome this limitation, we used CRISPR/Cas9-based editing to generate a human iPSC line expressing a fluorescent protein (mOrange) knocked-in at the last exon of the TH locus. After differentiation of the TH-mOrange reporter iPSC line, we confirmed that mOrange expression faithfully mimicked endogenous TH expression in iPSC-derived DAn. We also employed calcium imaging techniques to determine the intrinsic functional differences between dopaminergic and non-dopaminergic ventral midbrain neurons. Crucially, the brightness of mOrange allowed direct visualization of TH-expressing cells in heterogeneous cultures, and enabled us to isolate live mOrange-positive cells through fluorescence-activated cell sorting, for further differentiation. This technique, coupled to refined imaging and data processing tools, could advance the investigation of PD pathogenesis and might offer a platform to test potential new therapeutics for PD and other neurodegenerative diseases.

RevDate: 2020-10-15
CmpDate: 2020-10-15

Sakaguchi K, Yoneda M, Sakai N, et al (2019)

Comprehensive Experimental System for a Promising Model Organism Candidate for Marine Teleosts.

Scientific reports, 9(1):4948 pii:10.1038/s41598-019-41468-8.

A comprehensive experimental system for Japanese anchovy, a promising candidate model organism for marine teleosts, was established. Through the design of a rearing/spawning facility that controls the photoperiod and water temperature, one-cell eggs were continuously obtained shortly after spawning throughout the rearing period. The stages of eggs are indispensable for microinjection experiments, and we developed an efficient and robust microinjection system for the Japanese anchovy. Embryos injected with GFP mRNA showed strong whole-body GFP fluorescence and the survival rates of injected- and non-injected embryos were not significantly different, 87.5% (28 in 32 embryos) and 90.0% (45 in 50 embryos), respectively. We verified that the Tol2 transposon system, which mediates gene transfer in vertebrates, worked efficiently in the Japanese anchovy using the transient transgenesis protocol, with GFP or DsRed as the reporter gene. Finally, we confirmed that genome-editing technologies, namely Transcription Activator-Like Effector Nucleases (TALEN) and Clustered Regulatory Interspaced Short Palindromic Repeats (CRISPR)/Cas9, were applicable to the Japanese anchovy. In practice, specific gene-disrupted fishes were generated in the F1 generation. These results demonstrated the establishment of a basic, yet comprehensive, experimental system, which could be employed to undertake experiments using the Japanese anchovy as a model organism for marine teleost fish.

RevDate: 2020-10-15
CmpDate: 2020-10-15

Jeong YK, Yu J, S Bae (2019)

Construction of non-canonical PAM-targeting adenosine base editors by restriction enzyme-free DNA cloning using CRISPR-Cas9.

Scientific reports, 9(1):4939 pii:10.1038/s41598-019-41356-1.

Molecular cloning is an essential technique in molecular biology and biochemistry, but it is frequently laborious when adequate restriction enzyme recognition sites are absent. Cas9 endonucleases can induce site-specific DNA double-strand breaks at sites homologous to their guide RNAs, rendering an alternative to restriction enzymes. Here, by combining DNA cleavage via a Cas9 endonuclease and DNA ligation via Gibson assembly, we demonstrate a precise and practical DNA cloning method for replacing part of a backbone plasmid. We first replaced a resistance marker gene as a proof of concept and next generated DNA plasmids that encode engineered Cas9 variants (VQR, VRER and SpCas9-NG), which target non-canonical NGA, NGCG and NG protospacer-adjacent motif (PAM) sequences, fused with adenosine deaminases for adenine base editing (named VQR-ABE, VRER-ABE and NG-ABE, respectively). Ultimately, we confirmed that the re-constructed plasmids can successfully convert adenosine to guanine at endogenous target sites containing the non-canonical NGA, NGCG and NG PAMs, expanding the targetable range of the adenine base editing.

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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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CRISPR-Cas

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 @ gmail.com

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).

Timelines

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

Biographies

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 )