Highly efficient and safe genome editing by CRISPR-Cas12a using CRISPR RNA with a ribosyl-2'-O-methylated uridinylate-rich 3'-overhang in mouse zygotes.
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ABSTRACT:
SUBMITTER: Ha DI
PROVIDER: S-EPMC8080787 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Genome editing has been harnessed through the development of CRISPR system, and the CRISPR from Prevotella and Francisella 1 (Cpf1) system has emerged as a promising alternative to CRISPR-Cas9 for use in various circumstances. Despite the inherent multiple advantages of Cpf1 over Cas9, the adoption of Cpf1 has been unsatisfactory because of target-dependent insufficient indel efficiencies. Here, we report an engineered CRISPR RNA (crRNA) for highly efficient genome editing by Cpf1, which includes a 20-base target-complementary sequence and a uridinylate-rich 3'-overhang. When the crRNA is transcriptionally produced, crRNA with a 20-base target-complementary sequence plus a U4AU4 3'-overhang is the optimal configuration. U-rich crRNA also maximizes the utility of the AsCpf1 mutants and multiplexing genome editing using mRNA as the source of multiple crRNAs. Furthermore, U-rich crRNA enables a highly safe and specific genome editing using Cpf1 in human cells, contributing to the enhancement of a genome-editing toolbox.
| S-EPMC6128929 | biostudies-literature
Project description:The CRISPR/Cas9 system has been employed to efficiently edit the genomes of diverse model organisms. CRISPR-mediated mouse genome editing is typically accomplished by microinjection of Cas9 DNA/RNA and single guide RNA (sgRNA) into zygotes to generate modified animals in one step. However, microinjection is a technically demanding, labor-intensive, and costly procedure with poor embryo viability. Here, we describe a simple and economic electroporation-based strategy to deliver Cas9/sgRNA ribonucleoproteins into mouse zygotes with 100% efficiency for in vivo genome editing. Our methodology, designated as CRISPR RNP Electroporation of Zygotes (CRISPR-EZ), enables highly efficient and high-throughput genome editing in vivo, with a significant improvement in embryo viability compared with microinjection. Using CRISPR-EZ, we generated a variety of editing schemes in mouse embryos, including indel (insertion/deletion) mutations, point mutations, large deletions, and small insertions. In a proof-of-principle experiment, we used CRISPR-EZ to target the tyrosinase (Tyr) gene, achieving 88% bi-allelic editing and 42% homology-directed repair-mediated precise sequence modification in live mice. Taken together, CRISPR-EZ is simple, economic, high throughput, and highly efficient with the potential to replace microinjection for in vivo genome editing in mice and possibly in other mammals.
| S-EPMC4938170 | biostudies-literature
Project description:(1) Background: Gene editing technology, as represented by CRISPR is a powerful tool used in biomedical science. However, the editing efficiency of such technologies, especially in induced pluripotent stem cells (iPSCs) and other types of stem cells, is low which hinders its application in regenerative medicine; (2) Methods: A gene-editing system, COE, was designed and constructed based on CRISPR/Cas12a and Orip/EBNA1, and its editing efficiency was evaluated in human embryonic kidney 293T (HEK-293T) cells with flow cytometry and restriction fragment length polymorphism (RFLP) analysis. The COE was nucleofected into iPSCs, then, the editing efficiency was verified by a polymerase chain reaction and Sanger sequencing; (3) Results: With the extension of time, COE enables the generation of up to 90% insertion or deletion rates in HEK-293T cells. Furthermore, the deletion of a 2.5 kb fragment containing Exon 51 of the dystrophin gene (DMD) in iPSCs was achieved with high efficiency; out of 14 clones analyzed, 3 were positive. Additionally, the Exon 51-deleted iPSCs derived from cardiomyocytes had similar expression profiles to those of Duchenne muscular dystrophy (DMD) patient-specific iPSCs. Moreover, there was no residue of each component of the plasmid in the editing cells; (4) Conclusions: In this study, a novel, efficient, and safe gene-editing system, COE, was developed, providing a powerful tool for gene editing.
| S-EPMC8620414 | biostudies-literature
Project description:The ability to create targeted mutations using clustered regularly inter-spaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) 9 in support of forest tree biotechnology is currently limited. CRISPR/Cas12a is a novel CRISPR effector protein that not only broadens the CRISPR/Cas targeting range but also enables the generation of large-fragment deletions. In this study, a CRISPR/Cas12a system was evaluated for the induction of targeted mutations in the woody tree poplar (Populus alba × Populus glandulosa). Three Cas12a nucleases, namely, AsCas12a (Acidaminococcus sp. BV3L6), LbCas12a (Lachnospiraceae bacterium ND2006), and FnCas12a (Francisella tularensis subsp. novicidain U112), were used. We knocked out multiple targets of the phytoene desaturase gene 8 (PDS) using the CRISPR/Cas12a genome-targeting system, and the results indicated that the AsCas12a system is the most efficient. We further optimized the co-cultivation temperature after Agrobacterium-mediated transformation from 22 to 28°C to increase the Cas12a nuclease editing efficiency in poplar. AsCas12a showed the highest mutation efficiency, at 70%, and the majority of editing sites were composed of large-fragment deletions. By using this simple and high-efficiency CRISPR/Cas12a system, multiple targets can be modified to obtain multigene simultaneous knockout mutants in tree species, which will provide powerful tools with which to facilitate genetic studies of forest trees.
| S-EPMC7720674 | biostudies-literature
Project description: Not available
| S-EPMC10363544 | biostudies-literature
Project description:Electroporation of zygotes represents a rapid alternative to the elaborate pronuclear injection procedure for CRISPR/Cas9-mediated genome editing in mice. However, current protocols for electroporation either require the investment in specialized electroporators or corrosive pre-treatment of zygotes which compromises embryo viability. Here, we describe an easily adaptable approach for the introduction of specific mutations in C57BL/6 mice by electroporation of intact zygotes using a common electroporator with synthetic CRISPR/Cas9 components and minimal technical requirement. Direct comparison to conventional pronuclear injection demonstrates significantly reduced physical damage and thus improved embryo development with successful genome editing in up to 100% of living offspring. Hence, our novel approach for Easy Electroporation of Zygotes (EEZy) allows highly efficient generation of CRISPR/Cas9 transgenic mice while reducing the numbers of animals required.
| S-EPMC5933690 | biostudies-literature
Project description:Streptomyces strains produce a great number of valuable natural products. With the development of genome sequencing, a vast number of biosynthetic gene clusters with high potential for use in the discovery of valuable clinical drugs have been revealed. Therefore, emerging needs for tools to manipulate these biosynthetic pathways are presented. Although the clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas 9) system has exhibited great capabilities for gene editing in multiple Streptomyces strains, it has failed to work in some newly discovered strains and some important industrial strains. Additionally, the protospacer adjacent motif (PAM) recognition scope of this system sometimes limits its applications for generating precise site mutations and insertions. Here, we developed three efficient CRISPR-FnCas12a systems for multiplex genome editing in several Streptomyces strains. Each system exhibited advantages for different applications. The CRISPR-FnCas12a1 system was efficiently applied in the industrial strain Streptomyces hygroscopicus, in which SpCas9 does not work well. The CRISPR-FnCas12a2 system was used to delete large fragments ranging from 21.4 to 128 kb. Additionally, the CRISPR-FnCas12a3 system employing the engineered FnCas12a mutant EP16, which recognizes a broad spectrum of PAM sequences, was used to precisely perform site mutations and insertions. The CRISPR-FnCas12a3 system addressed the limitation of TTN PAM recognition in Streptomyces strains with high GC contents. In summary, all the CRISPR-FnCas12a systems developed in this study are powerful tools for precise and multiplex genome editing in Streptomyces strains.
| S-EPMC7338789 | biostudies-literature
Project description:Genome editing tools such as the clustered regularly interspaced short palindromic repeat (CRISPR)-associated system (Cas) have been widely used to modify genes in model systems including animal zygotes and human cells, and hold tremendous promise for both basic research and clinical applications. To date, a serious knowledge gap remains in our understanding of DNA repair mechanisms in human early embryos, and in the efficiency and potential off-target effects of using technologies such as CRISPR/Cas9 in human pre-implantation embryos. In this report, we used tripronuclear (3PN) zygotes to further investigate CRISPR/Cas9-mediated gene editing in human cells. We found that CRISPR/Cas9 could effectively cleave the endogenous β-globin gene (HBB). However, the efficiency of homologous recombination directed repair (HDR) of HBB was low and the edited embryos were mosaic. Off-target cleavage was also apparent in these 3PN zygotes as revealed by the T7E1 assay and whole-exome sequencing. Furthermore, the endogenous delta-globin gene (HBD), which is homologous to HBB, competed with exogenous donor oligos to act as the repair template, leading to untoward mutations. Our data also indicated that repair of the HBB locus in these embryos occurred preferentially through the non-crossover HDR pathway. Taken together, our work highlights the pressing need to further improve the fidelity and specificity of the CRISPR/Cas9 platform, a prerequisite for any clinical applications of CRSIPR/Cas9-mediated editing.
| S-EPMC4417674 | biostudies-literature
Project description:CRISPR-Cas9 has been widely adopted as the basic toolkit for precise genome-editing and engineering in various organisms. Alternative to Cas9, Cas12 or Cpf1 uses a simple crRNA as a guide and expands the protospacer adjacent motif (PAM) sequence to TTTN. This unique PAM sequence of Cpf1 may significantly increase the on-target editing efficiency due to lower chance of Cpf1 misreading the PAMs on a high GC genome. To demonstrate the utility of CRISPR-Cpf1, we have optimized the CRISPR-Cpf1 system and achieved high-editing efficiency for two counter-selectable markers in the industrially-relevant oleaginous yeast Yarrowia lipolytica: arginine permease (93% for CAN1) and orotidine 5'-phosphate decarboxylase (~96% for URA3). Both mutations were validated by indel mutation sequencing. For the first time, we further expanded this toolkit to edit three sulfur house-keeping genetic markers (40%-75% for MET2, MET6 and MET25), which confers yeast distinct colony color changes due to the formation of PbS (lead sulfide) precipitates. Different from Cas9, we demonstrated that the crRNA transcribed from a standard type II RNA promoter was sufficient to guide Cpf1 endonuclease activity. Furthermore, modification of the crRNA with 3' polyUs facilitates the faster maturation and folding of crRNA and improve the genome editing efficiency. We also achieved multiplexed genome editing, and the editing efficiency reached 75%-83% for duplex genomic targets (CAN1-URA3 and CAN1-MET25) and 41.7% for triplex genomic targets (CAN1-URA3-MET25). Taken together, this work expands the genome-editing toolbox for oleaginous yeast species and may accelerate our ability to engineer oleaginous yeast for both biotechnological and biomedical applications.
| S-EPMC6906711 | biostudies-literature
Project description:The rapid-growing and genetically tractable methanogen Methanococcus maripaludis is a promising host organism for the biotechnological conversion of carbon dioxide and renewable hydrogen to fuels and value-added products. Expansion of its product scope through metabolic engineering necessitates reliable and efficient genetic tools, particularly for genome edits that affect the primary metabolism and cell growth. Here, we have designed a genome-editing toolbox by utilizing Cas12a from Lachnospiraceae bacterium ND2006 (LbCas12a) in combination with the homology-directed repair machinery endogenously present in M. maripaludis. This toolbox can delete target genes with a success rate of up to 95%, despite the hyperpolyploidy of M. maripaludis. For the purpose of demonstrating a large deletion, the M. maripaludis flagellum operon (∼8.9 kbp) was replaced by the Escherichia coli β-glucuronidase gene. To facilitate metabolic engineering and flux balancing in M. maripaludis, the relative strength of 15 different promoters was quantified in the presence of two common growth substrates, either formate or carbon dioxide and hydrogen. This CRISPR/LbCas12a toolbox can be regarded as a reliable and quick method for genome editing in a methanogen.
| S-EPMC9295151 | biostudies-literature