Gene Editing By CRISPR/Cas9

Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated proteins (Cas) systems, found in bacteria and archaea, carry out the adaptive immune responses against invading genetic elements (virus or plasmid) by using RNAs to guide site-specific cleavage of genetic material. Type II of these systems, using an RNA-guided nuclease (Cas9) to destroy invading DNA, was demonstrated that it can efficiently cleave of any given DNA sequence in tube. We adapted this system in higher eukaryotes by synthesis the human-codon optimized Cas9and demonstrated that it could site-specifically cut eukaryote DNA in zebrafish embryos by co-injection of Cas9mRNA and chimeric RNA. We added a NLS-flag-linker fragment to N terminal of Cas9, which enhanced CRISPR/Cas9activity and successfully localized Cas9to nuclei of mammalian cells. Via embryo microinjection of the modified Cas9mRNA and pre-annealed chimeric RNA, we successfully achieved endogenous gene modification of mice for the first time. Then, taking the advantage of the multiplexable genome engineering feature of the CRISPR/Cas9system, we generated multiple gene modifications mice and rat by embryo co-microinjection of Cas9mRNA and multiple sgRNAs. Encouraged by our successes in CRISPR/Cas9-mediated gene targeting, we extended the application of the CRISPR/Cas9system to multiplex genetic engineering in one cell-stage embryos of cynomolgus monkeys and successfully obtained founder animals harboring two gene modifications. Our results demonstrated CRISPR/Cas9is a RNA-based new class of genome engineering approach which works well in mouse, rat and cynomolgus monkey. In our following study, we found that co-microinjection of mouse embryos with Cas9mRNA and single guide RNA (sgRNA) induced on-target and off-target mutations that were transmissible to offspring. To improve the specificity of the system, we were therefore interested in exploring the use of CRISPR/Cas9nickases for in vivo genome editing. Because nicked genomic DNA is corrected by the endogenous base-excision repair pathway, Cas9nickases would be expected to induce little or no damage to the genome. However, if single stranded nicks are located close together on opposite DNA strands, then the resulting double stranded break may be imprecisely repaired by NHEJ. Thus, co-expression of Cas9nickases and sgRNAs to closely paired sites on opposing DNA strands provides a general strategy for genome modification with minimal off-target damage. By staining of y-H2AX in transfected cells, Cas9nickases and paired sgRNA indeed reduced, but not eliminate, potential off-target damage resulting from NHEJ at double-stranded breaks. In mouse, Cas9nickase and paired sgRNAs can be used to efficiently mutate genes without detectable damage at known off-target sites. To further assess the specificity of the paired nickase strategy, we assayed24known off-target sites in Hela cells. T7EN cleavage assay and deep sequencing of the samples confirmed a very low frequency of indels, indistinguishable from background, in cells treated with Cas9nickase and paired sgRNAs. To define the orientation and distance between paired sgRNA for effective damage by NHEJ, we selected a set of nested target sites on opposing strands in exon two of the human RAG1gene. The results showed maximal cleavage was observed at sites oriented tail-to-tail and separated by10to+30bp. We also applied the strategy to in vivo modification of multiple genes and demonstrate that a multiplex paired nickase strategy is effective for modification of multiple genes in vivo and generation of sizeable deletions of genomic DNA. In both mouse embyros and in cultured cells, we find no distinct evidence for NHEJ-induced damage by Cas9nickase at off-target sites prone to mutation by wild-type Cas9endonuclease. Therefore, the use of Cas9nickase and paired sgRNAs greatly increases the fidelity of the Cas9system for in vivo genome editing without compromising efficiency. Our method is applicable for genome editing of any model organism and minimizes the confounding problems of off-target mutations.