MOE Key Laboratory of Bioinformatics and Bioinformatics Division, Center for Synthetic and System Biology, Department of Automation, Beijing National Research Center for Information Science and Technology, Tsinghua University, Beijing, 100084, China.
Nat Commun. 2019 Feb 4;10(1):560. doi: 10.1038/s41467-019-08395-8.
Although Cas9 nucleases are remarkably diverse in microorganisms, the range of genomic sequences targetable by a CRISPR/Cas9 system is restricted by the requirement of a short protospacer adjacent motif (PAM) at the target site. Here, we generate a group of chimeric Cas9 (cCas9) variants by replacing the key region in the PAM interaction (PI) domain of Staphylococcus aureus Cas9 (SaCas9) with the corresponding region in a panel of SaCas9 orthologs. By using a functional assay at target sites with different nucleotide recombinations at PAM position 3-6, we identify several cCas9 variants with expanded recognition capability at NNVRRN, NNVACT, NNVATG, NNVATT, NNVGCT, NNVGTG, and NNVGTT PAM sequences. In summary, we provide a panel of cCas9 variants accessible up to 1/4 of all the possible genomic targets in mammalian cells.
尽管 Cas9 核酸酶在微生物中种类繁多，但 CRISPR/Cas9 系统可靶向的基因组序列范围受到目标位点短的前导间隔相邻基序 (PAM) 的要求限制。在这里，我们通过用一组金黄色葡萄球菌 Cas9 (SaCas9) 同源物的相应区域替换 PAM 相互作用 (PI) 结构域中的关键区域，生成了一组嵌合 Cas9 (cCas9) 变体。通过在 PAM 位置 3-6 处具有不同核苷酸重组的靶位点进行功能测定，我们鉴定了几种 cCas9 变体，它们在 NNVRRN、NNVACT、NNVATG、NNVATT、NNVGCT、NNVGTG 和 NNVGTT PAM 序列中具有扩展的识别能力。总之，我们提供了一组 cCas9 变体，可用于哺乳动物细胞中多达四分之一的所有可能的基因组靶标。
