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.

蛋白质的位点特异性化学缀合可以增强其治疗和诊断效用，但很少应用于 CRISPR-Cas9，这是一个快速发展的领域，具有巨大的治疗潜力。同源定向修复的低效率仍然是 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，我们开发了将寡核苷酸位点特异性缀合到重组 Cas9 蛋白的方法，该重组 Cas9 蛋白含有具有正交化学反应性的遗传编码非规范氨基酸。Cas9-寡核苷酸偶联物通过碱基配对将未修饰的供体 DNA 模板募集到靶位点，显着提高了人类细胞培养和小鼠受精卵中的同源定向修复效率。