Nakagawa Ryoya, Ishiguro Soh, Okazaki Sae, Mori Hideto, Tanaka Mamoru, Aburatani Hiroyuki, Yachie Nozomu, Nishimasu Hiroshi, Nureki Osamu
Department of Biological Sciences, Graduate School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-0033, Japan.
School of Biomedical Engineering, Faculty of Applied Science and Faculty of Medicine, The University of British Columbia, Vancouver, British Columbia, Canada.
Commun Biol. 2022 Mar 8;5(1):211. doi: 10.1038/s42003-022-03149-7.
The RNA-guided DNA endonuclease Cas9 is a versatile genome-editing tool. However, the molecular weight of the commonly used Streptococcus pyogenes Cas9 is relatively large. Consequently, its gene cannot be efficiently packaged into an adeno-associated virus vector, thereby limiting its applications for therapeutic genome editing. Here, we biochemically characterized the compact Cas9 from Campylobacter jejuni (CjCas9) and found that CjCas9 has a previously unrecognized preference for the NVRYAC protospacer adjacent motif. We thus rationally engineered a CjCas9 variant (enCjCas9), which exhibits enhanced cleavage activity and a broader targeting range both in vitro and in human cells, as compared with CjCas9. Furthermore, a nickase version of enCjCas9, but not CjCas9, fused with a cytosine deaminase mediated C-to-T conversions in human cells. Overall, our findings expand the CRISPR-Cas toolbox for therapeutic genome engineering.
RNA引导的DNA内切酶Cas9是一种多功能的基因组编辑工具。然而，常用的化脓性链球菌Cas9分子量相对较大。因此，其基因不能有效地包装到腺相关病毒载体中，从而限制了其在治疗性基因组编辑中的应用。在此，我们对空肠弯曲菌的紧凑型Cas9（CjCas9）进行了生化特性分析，发现CjCas9对NVRYAC原间隔相邻基序有此前未被认识到的偏好。因此，我们合理设计了一种CjCas9变体（enCjCas9），与CjCas9相比，它在体外和人类细胞中均表现出增强的切割活性和更广泛的靶向范围。此外，与胞嘧啶脱氨酶融合的enCjCas9切口酶版本（而非CjCas9）在人类细胞中介导了C到T的转化。总体而言，我们的研究结果扩展了用于治疗性基因组工程的CRISPR-Cas工具库。
