Improving the efficiency of precise genome editing with site-specific Cas9-oligonucleotide conjugates
Improving the efficiency of precise genome editing with site-specific Cas9-oligonucleotide conjugates
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利用位点特异性 Cas9-寡核苷酸缀合物提高精确基因组编辑的效率
DOI：
10.1126/sciadv.aaz0051
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发表时间：
2020-04-01
期刊：
影响因子：
13.6
通讯作者：
Liu, Tao
中科院分区：
文献类型：
--
作者：
Ling, Xinyu;Xie, Bingteng;Liu, Tao
一种化学蛋白质-DNA结合方法增强了人类细胞和小鼠受精卵的精确基因组编辑。蛋白质的定点化学结合可以提高其治疗和诊断的效用，但很少被应用于CRISPR-Cas9，这是一个快速增长的领域，具有巨大的治疗潜力。同源定向修复效率低仍然是CRISPR-Cas9介导的精确基因组编辑的主要障碍，这受到切割位点供体DNA模板浓度低的限制。在这项研究中，我们发展了一种方法，将寡核苷酸与含有具有正交化学反应活性的遗传编码的非规范氨基酸的重组Cas9蛋白定点连接。Cas9-寡核苷酸结合物通过碱基配对将未经修饰的供体DNA模板招募到目标位置，显著提高了人类细胞培养和小鼠受精卵的同源定向修复效率。这些化学修饰的Cas9突变体提供了一个补充化学修饰的核酸的额外工具，用于提高基于CRISPR-Cas9的基因组编辑系统的效用。
A chemical protein-DNA conjugation approach enhances precise genome editing in human cells and mouse zygotes. Site-specific chemical conjugation of proteins can enhance their therapeutic and diagnostic utility but has seldom been applied to CRISPR-Cas9, which is a rapidly growing field with great therapeutic potential. The low efficiency of homology-directed repair remains a major hurdle in CRISPR-Cas9–mediated precise genome editing, which is limited by low concentration of donor DNA template at the cleavage site. In this study, we have developed methodology to site-specifically conjugate oligonucleotides to recombinant Cas9 protein containing a genetically encoded noncanonical amino acid with orthogonal chemical reactivity. The Cas9-oligonucleotide conjugates recruited an unmodified donor DNA template to the target site through base pairing, markedly increasing homology-directed repair efficiency in both human cell culture and mouse zygotes. These chemically modified Cas9 mutants provide an additional tool, one that is complementary to chemically modified nucleic acids, for improving the utility of CRISPR-Cas9–based genome-editing systems.