Institute of Pharmacology and Toxicology, University of Zurich, Zurich, Switzerland.
Institute of Molecular Health Sciences, ETH Zurich, Zurich, Switzerland.
Nat Chem Biol. 2024 Mar;20(3):333-343. doi: 10.1038/s41589-023-01427-x. Epub 2023 Sep 21.
CRISPR-Cas9 genome engineering is a powerful technology for correcting genetic diseases. However, the targeting range of Cas9 proteins is limited by their requirement for a protospacer adjacent motif (PAM), and in vivo delivery is challenging due to their large size. Here, we use phage-assisted continuous directed evolution to broaden the PAM compatibility of Campylobacter jejuni Cas9 (CjCas9), the smallest Cas9 ortholog characterized to date. The identified variant, termed evoCjCas9, primarily recognizes NAH and NHA PAM sequences, which occur tenfold more frequently in the genome than the canonical NVRYAC PAM site. Moreover, evoCjCas9 exhibits higher nuclease activity than wild-type CjCas9 on canonical PAMs, with editing rates comparable to commonly used PAM-relaxed SpCas9 variants. Combined with deaminases or reverse transcriptases, evoCjCas9 enables robust base and prime editing, with the small size of evoCjCas9 base editors allowing for tissue-specific installation of A-to-G or C-to-T transition mutations from single adeno-associated virus vector systems.
CRISPR-Cas9 基因组工程是一种强大的纠正遗传疾病的技术。然而，Cas9 蛋白的靶向范围受到其对前导间隔相邻基序 (PAM) 的要求限制，并且由于其尺寸较大，体内递送具有挑战性。在这里，我们使用噬菌体辅助连续定向进化来扩大迄今为止表征的最小 Cas9 同源物 Campylobacter jejuni Cas9 (CjCas9) 的 PAM 兼容性。鉴定出的变体称为 evoCjCas9，主要识别 NAH 和 NHA PAM 序列，这些序列在基因组中的出现频率比典型的 NVRYAC PAM 位点高十倍。此外，与常见的 PAM 宽松 SpCas9 变体相比，eVoCjCas9 在典型的 PAMs 上表现出更高的核酸酶活性，编辑效率相当。与脱氨酶或逆转录酶结合使用时，eVoCjCas9 能够实现强大的碱基编辑和 Prime 编辑，eVoCjCas9 的小尺寸碱基编辑器允许从单个腺相关病毒载体系统对组织特异性安装 A 到 G 或 C 到 T 的转换突变。
