一种新型的CRISPR/Cas9-hLacI双链DNA供体适配基因编辑系统

A novel CRISPR/Cas9-hLacI donor adapting system for dsDNA-templated gene editing

摘要:

在CRISPR/Cas9系统介导的基因编辑中，借助于双链DNA （double-stranded DNA，dsDNA）供体模板的重组效应能够实现对目标基因组靶位点的精确编辑和基因敲入，然而高等真核生物细胞中同源重组的低效性限制了该基因编辑策略的发展和应用。为提高CRISPR/Cas9系统介导dsDNA供体模板的同源重组效率，本研究利用大肠杆菌（Escherichia coli）乳糖操纵子阻遏蛋白LacI与操纵序列LacO特异性结合的特点，通过重组DNA技术将密码子人源化优化的阻遏蛋白基因LacI分别与脓链球菌（Streptococcus pyogenes）源的SpCas9和路邓葡萄球菌（Staphylococcus lugdunensis）源的SlugCas9-HF融合表达，通过PCR将操纵序列LacO与dsDNA供体嵌合，构建了新型的CRISPR/Cas9-hLacI供体适配系统（donor adapting system，DAS）。首先在报告载体水平上对Cas9核酸酶活性、DAS介导的同源引导修复（homology-directed repair，HDR）效率进行了验证和优化，其次在基因组水平对其介导的基因精确编辑进行了检测，并最终利用CRISPR/SlugCas9-hLacI DAS在HEK293T细胞中实现了VEGFA位点的精确编辑，效率高达30.5%，显著高于野生型。综上所述，本研究开发了新型的CRISPR/Cas9-hLacI供体适配基因编辑系统，丰富了CRISPR/Cas9基因编辑技术种类，为以后的基因编辑及分子设计育种研究提供了新的工具。

关键词:基因编辑;CRISPR/Cas9;LacI基因;LacO序列;供体适配

Abstract:

During the gene editing process mediated by CRISPR/Cas9, precise genome editing and gene knock-in can be achieved by the homologous recombination of double-stranded DNA (dsDNA) donor template. However, the low-efficiency of homologous recombination in eukaryotic cells hampers the development and application of this gene editing strategy. Here, we developed a novel CRISPR/Cas9-hLacI donor adapting system (DAS) to enhance the dsDNA-templated gene editing, taking the advantage of the specific binding of the LacI repressor protein and the LacO operator sequence derived for the Escherichia coli lactose operon. The codon-humanized LacI gene was fused as an adaptor to the Streptococcus pyogenes Cas9 (SpCas9) and Staphylococcus lugdunensis Cas9 (SlugCas9-HF) genes, and the LacO operator sequence was used as the aptamer and linked to the dsDNA donor template by PCR. The Cas9 nuclease activity after the fusion and the homology-directed repair (HDR) efficiency of the LacO-linked dsDNA template were firstly examined using surrogate reporter assays with the corresponding reporter vectors. The CRISPR/Cas9-hLacI DASs mediated genome precise editing were further checked, and we achieved a high efficiency up to 30.5% of precise editing at the VEGFA locus in HEK293T cells by using the CRISPR/SlugCas9-hLacI DAS. In summary, we developed a novel CRISPR/Cas9-hLacI DAS for dsDNA-templated gene editing, which enriches the CRISPR/Cas9-derived gene editing techniques and provides a novel tool for animal molecular design breeding researches.

Key words:gene editing;CRISPR/Cas9;LacI gene;LacO sequence;donor adapting