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Found: 26 168
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Improving the Efficiency of CRISPR/Cas9-Mediated Non-Homologous End Joining Gene Knockout Using Small Molecules in Porcine Cells.
The CRISPR/Cas9 genome editing system has emerged as an effective platform to generate loss-of-function gene edits through non-homologous end joining (NHEJ) without a repair template. To verify whether small molecules can enhance the efficiency of CRISPR/ Cas9-mediated NHEJ gene editing in porcine cells, this experiment investigated the effects of six small-molecule compounds, namely Repsox, Zidovudine, IOX1, GSK-J4, YU238259, and GW843682X, on the efficiency of CRISPR/Cas9-mediated NHEJ gene editing. The results showed the optimal concentrations of the small molecules, including Repsox, Zidovudine, IOX1, GSK-J4, YU238259, and GW843682X, for in vitro-cultured PK15 viability. Compared with the control group, the single small molecules Repsox, Zidovudine, GSK-J4, and IOX1 increased the efficiency of NHEJ-mediated gene editing 3.16-fold, 1.17-fold, 1.16-fold, and 1.120-fold, respectively, in the Cas9-sgRNA RNP delivery system. There were no benefits when using YU238259 and GW843682X compared with the control group. In the CRISPR/Cas9 plasmid delivery system, the Repsox, Zidovudine, IOX1, and GSK-J4 treatments increased the efficiency of NHEJ-mediated gene editing 1.47-fold, 1.15-fold, 1.21-fold, and 1.23-fold, respectively, compared with the control group. Repsox can also improve the efficiency of NHEJ-mediated multi-gene editing based on a CRISPR sgRNA-tRNA array. We also explored the mechanism of Repsox's effect on the efficiency of NHEJ-mediated gene editing. The results showed that Repsox reduces the expression levels of SMAD2, SMAD3, and SMAD4 in the TGF-β pathway, indicating that Repsox can increase the efficiency of CRISPR NHEJ-mediated gene editing in porcine cells through the TGF-β pathway.
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Editing nature: Local roots of global governance.
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Rapid Generation of Human Genetic Loss-of-Function iPSC Lines by Simultaneous Reprogramming and Gene Editing.
Specifically ablating genes in human induced pluripotent stem cells (iPSCs) allows for studies of gene function as well as disease mechanisms in disorders caused by loss-of-function (LOF) mutations. While techniques exist for engineering such lines, we have developed and rigorously validated a method of simultaneous iPSC reprogramming while generating CRISPR/Cas9-dependent insertions/deletions (indels). This approach allows for the efficient and rapid formation of genetic LOF human disease cell models with isogenic controls. The rate of mutagenized lines was strikingly consistent across experiments targeting four different human epileptic encephalopathy genes and a metabolic enzyme-encoding gene, and was more efficient and consistent than using CRISPR gene editing of established iPSC lines. The ability of our streamlined method to reproducibly generate heterozygous and homozygous LOF iPSC lines with passage-matched isogenic controls in a single step provides for the rapid development of LOF disease models with ideal control lines, even in the absence of patient tissue.
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Establishment of a Novel CRISPR/Cas9-Based Multiplex Editing System in the Citrus Postharvest Pathogen Penicillium digitatum.
Penicillium digitatum, the causal fungus of the citrus green mold, leads to substantial postharvest losses in the citrus industry. In this study, we engineered a versatile CRISPR/Cas9-mediated gene editing platform capable of generating multiple sgRNAs from a single polycistronic transcript. By incorporating endogenous tRNAs and the strong promoter PdMLE1 into the CRISPR/Cas9 system, the efficiency of single gene editing can reach up to 94.2%. By taking advantage of the efficient shearing and processing capabilities of tRNA, the platform enabled multiplex editing with efficiencies of 44.4% for two-gene and 33.3% for three-gene modifications, respectively. After two rounds of three-gene editing, we were able to successfully obtain hextuple-gene mutants. Finally, functional characterization revealed that the target polysaccharide-lyase-encoding genes play limited roles in pathogenicity in P. digitatum. Taken together, our results represent a powerful tool for genome engineering in P. digitatum, facilitating research into its pathogenesis.
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Prime Editing: A Novel Cas9-Reverse Transcriptase Fusion May Revolutionize Genome Editing.
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CRISPR/Cas9-based genome editing of 14 lipid metabolic genes reveals a sporopollenin metabolon ZmPKSB-ZmTKPR1-1/-2 required for pollen exine formation in maize.
Lipid biosynthesis and transport are essential for plant male reproduction. Compared with Arabidopsis and rice, relatively fewer maize lipid metabolic genic male-sterility (GMS) genes have been identified, and the sporopollenin metabolon in maize anther remains unknown. Here, we identified two maize GMS genes, ZmTKPR1-1 and ZmTKPR1-2, by CRISPR/Cas9 mutagenesis of 14 lipid metabolic genes with anther stage-specific expression patterns. Among them, tkpr1-1/-2 double mutants displayed complete male sterility with delayed tapetum degradation and abortive pollen. ZmTKPR1-1 and ZmTKPR1-2 encode tetraketide α-pyrone reductases and have catalytic activities in reducing tetraketide α-pyrone produced by ZmPKSB (polyketide synthase B). Several conserved catalytic sites (S128/130, Y164/166 and K168/170 in ZmTKPR1-1/-2) are essential for their enzymatic activities. Both ZmTKPR1-1 and ZmTKPR1-2 are directly activated by ZmMYB84, and their encoded proteins are localized in both the endoplasmic reticulum and nuclei. Based on protein structure prediction, molecular docking, site-directed mutagenesis and biochemical assays, the sporopollenin biosynthetic metabolon ZmPKSB-ZmTKPR1-1/-2 was identified to control pollen exine formation in maize anther. Although ZmTKPR1-1/-2 and ZmPKSB formed a protein complex, their mutants showed different, even opposite, defective phenotypes of anther cuticle and pollen exine. Our findings discover new maize GMS genes that can contribute to male-sterility line-assisted maize breeding and also provide new insights into the metabolon-regulated sporopollenin biosynthesis in maize anther.
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Online Databases of Genome Editing in Cardiovascular and Metabolic Diseases.
Metabolic and cardiovascular diseases are world-concerning pathologies that affect an important percentage of the population. Nowadays, advances in the genetic background of these diseases allow new approaches to models and therapies, as well as different gene edition trials. Furthermore, technological improvements in gene editing go along with the development of new online and biocomputational tools that provide us alternative ways to explore pathologies. In this chapter, historical gene editing methods are discussed but focusing on CRISPR-Cas system in detail and also online resources available to perform these types of experiments. Here, the different strategies for gene editing and their online tools are gathered, putting the light on its application in the study and treatment of cardiovascular and metabolic diseases.
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Genome Editing Mediated by Primordial Germ Cell in Chicken.
Genome editing technology has facilitated the studies on exploring specific gene functions in diverse living organisms. The technology has also contributed to creating high-value livestock in industry fields in terms of enhancing productivity or acquiring disease resistance. Particularly, applying genome editing technologies in avian species has been emphasized in both academic and industrial fields due to their unique developmental patterns as well as application possibilities. To accomplish genome editing in avian species, gene integration into chicken primordial germ cell (PGC) genome using a virus or transposition systems has been widely used, and recently developed programmable genome editing technologies including clustered regularly interspaced short palindromic repeat (CRISPR) and CRISPR-associated (Cas9) systems enable to edit the genetic information precisely for maximizing the application potentials of avian species. In these regards, this chapter will cover the methods for producing genome-edited chickens, particularly by CRISPR/Cas9 technologies allowing targeted gene insertion, gene knockout, and gene tagging.
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In situ gene editing of hematopoietic stem cells via AAV-delivered CRISPR guide RNAs.
Hematopoietic stem cells (HSCs) are self-renewing, multipotent, and engraftable precursors of all blood cells. Efficient delivery of therapeutic gene products and gene editing machinery to correct disease-causing gene variants in endogenous HSCs while they remain in the body holds exciting potential to leverage HSC potency for the treatment of monogenic blood disorders. Toward this goal, we used adeno-associated virus (AAV) to deliver CRISPR guide RNAs (gRNAs) to edit HSC genomes in situ in Ai9;SpCas9-EGFP transgenic mice carrying a Cas9-activatable Lox-STOP-Lox-tdTomato reporter cassette together with a constitutive SpCas9-2A-EGFP. Using a variety of conditions and vector designs, we tested whether systemic administration to these mice of AAVs carrying SpCas9-compatible gRNAs designed to cut DNA upstream and downstream of the STOP cassette would induce tdTomato expression in HSCs. Our findings identify self-complementary AAVs (scAAVs) and increased ratio of guide to Cas9 as parameters facilitating higher editing efficiency. Of note, we find preserved multilineage output and engraftability of HSCs upon scAAV-gRNA editing. In an example application of this technology, we explore the potential for in situ HSC gene editing by dual AAV-CRISPR delivery and demonstrate robust gene modification, concurrent with induction of therapeutic fetal hemoglobin, in a sickle cell disease mouse model modified to express SpCas9. In summary, this work offers a sensitive and adaptable platform that allows robust modification of HSC genomes in situ.
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CRISPR-Cas9 Genome Editing in Auxotrophic and Non-auxotrophic Fission Yeast Strains.
The CRISPR/Cas system is a very powerful genome-editing tool that has been developed over the past decade to optimize genome editing for many organisms. Here, we describe a rapid genome-editing method for fission yeast using the CRISPR-Cas9 system. It allows rapid generation of desired auxotrophic and non-auxotrophic strains without perturbing the local genome content by avoiding the insertion of selection markers at target loci.
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