快速构建CRISPR/Cas9基因组靶向编辑系统

CRISPR/Cas9核酸酶作为一种新的基因组靶向编辑技术,已成功应用于多种动植物基因组修饰研究.CRISPR/Cas9作用后的阳性细胞筛选和富集是该技术的关键之一.本研究以鸡EAVHP(endogenous avian retrovirus-HP)基因和MSTN(myostatin)基因为例,从靶位点的选择、表达载体构建、双基因报告载体构建和核酸酶活性验证4个方面,系统研究了CRISPR/Cas9核酸酶技术平台.结果表明,利用寡聚核苷酸直接退火方法,构建表达载体和报告载体的阳性率分别高达100%和89.5%.报告载体的PuroR(puromycin resistant gene)和e GFP(enhanced green fluorescent protein)基因的成功表达表明,构建的CRISPR/Cas9系统能有效切割靶序列,并用于后续阳性克隆的筛选和富集.本方法摒弃了传统分子克隆的PCR扩增和酶切处理目标基因的方法,而是利用寡聚核苷酸直接退火获得含有黏性末端的目标DNA,简化了载体构建过程,低成本且快速获得CRISPR/Cas9基因组靶向编辑系统.

Rapid Construction of CRISPR/Cas9 System for Target Genomic Editing

As a novel technology for genome targeted modification, CRISPR/Cas9 nucleases are applied to successfully edit genomes in various species. However, screening and enrichment of CRISPR/Cas9-editing positive clones is a key issue of this technology. In this study, we explored a specific CRISPR/Cas9 system targeting chicken EAV-HP (endogenous avian retrovirus-HP) and MSTN (myostatin) genes. The CRISPR/Cas9 platform includes target site selection, expression vectors cloning, dual-gene reporter vector construction and nuclease activity validation. The results demonstrated that the efficiency of clone strategy with oligonucleotides direct annealing was up to 100% and 89.5% in construction of expression vectors and reporter vectors, respectively. Reporter genes PuroR (puromycin resistant gene) and eGFP (enhanced green fluorescent protein) were restored in 293T cells. Thus this result suggested that the designed CRISPR/Cas9 could cut target sequences efficiently, which guaranteed further use for target gene editing. To simplify CRISPR/Cas9 vectors construction, target DNA fragments harboring sticky ends were achieved via oligonucleotides annealing without traditional procedures of PCR and double digestion. In summary, we provide a feasible and time-saving approach to construct CRISPR/Cas9 system rapidly.