【作者】 沈彬；

【导师】 黄行许；

【作者基本信息】 南京大学 ， 生物学， 2014， 博士

【摘要】 CRISPR/Cas系统作为细菌和古细菌的获得性免疫系统,通过RNA介导,特异性的切割外源遗传物质,用以对抗入侵的病毒和质粒。利用Cas9来摧毁入侵DNA的Type Ⅱ CRISPR/Cas系统,被证明可以在试管中高效切割任一给定的DNA。我们通过密码子优化,合成Cas9基因,通过注射Cas9mRNA和chiRNA到斑马鱼受精卵,发现Cas9可以在斑马鱼受精卵中切割真核基因。在Cas9N端加上NLS-flag-linker,可以更好的让Cas9进入细胞核。通过注射NLS-flag-linker Cas9mRNA和chiRNA到小鼠—细胞其受精卵,我们成功得到首只基因修饰小鼠,证明了CRISPR/Cas9可以应用于小鼠的基因操作。然后利用CRISPR/Cas9的多能性,通过同时注射Cas9mRNA和多个sgRNA,我们得到多基因修饰的小鼠和大鼠。受到这些成功的鼓励,我们扩展了CRISPR/Cas9的使用范围,通过在食蟹猴—细胞期受精卵中注射Cas9mRNA和sgRNA,我们成功得到两只含有Ppra-γ和RagI突变的食蟹猴。在随后的研究中,我们发现在小鼠体内,CRISPR/Cas9会存在脱靶效应,并且脱靶位点的突变也可以遗传到后代。为了提高CRISPR/Cas9的特异性,我们尝试使用Cas9切口酶和一对sgRNA,两个相近的切口可以组成DNA双链断裂,诱导细胞发生非同源末端连接修复,引入突变,而在脱靶位点几乎只会形成一个切口,但是基因组上的切口可以通过碱基切除修复方式完美修复。通过转染细胞的γ-H2AX染色,我们发现这种策略(Cas9切口酶/sgRNA对)可以降低对基因组的损伤。在小鼠上,这种策略可以高效突变目的基因,但是检测不到已知的脱靶位点。在Hela细胞上,我们检测24个已知脱靶位点,通过T7EN和深度测序分析,Cas9切口酶/sgRNA对确实可以大大降低脱靶效应。通过系列成对sgRNA的切割实验,我们发现相间10-30bp、尾对尾方式排列的成对sgRNA具有高效的切割活性。利用这种策略,我们在小鼠体内可以同时敲除多个基因,并且可以进行大片段敲除。在细胞和小鼠上,我们都证明了Cas9切口酶/sgRNA对可以降低脱靶效应,并且不影响靶位点突变效率,这种方法可以广泛应用到其他物种的基因打靶。更多还原

【Abstract】 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.更多还原