School of Ophthalmology and Optometry, Eye Hospital, Wenzhou Medical University, State Key Laboratory and Key Laboratory of Vision Science, Ministry of Health and Zhejiang Provincial Key Laboratory of Ophthalmology and Optometry, Wenzhou, Zhejiang, China.
Reproduction Center, Department of Obstetrics and Gynecology, The Second Affiliated Hospital and Yuying Children's Hospital of Wenzhou Medical University, Wenzhou, Zhejiang, China.
Gene Ther. 2022 Aug;29(7-8):458-463. doi: 10.1038/s41434-022-00319-4. Epub 2022 Jan 31.
CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated endonuclease Cas9) nucleases have been widely applied for genome engineering. Staphylococcus aureus Cas9 (SaCas9) is compact, which can be packaged in AAV (adeno-associated virus) vector for in vivo gene editing. While, wild-type SaCas9 can induce unwanted off-target mutations and substantially limits the applications. So far, there are two reported SaCas9 variants with high-fidelity, including efSaCas9 from our previous study and SaCas9-HF. However, it remains unknown which one possessing the better fidelity and higher activity. Here, we performed a parallel comparison of efSaCas9 and SaCas9-HF in human cells through fluorescent reporter system and target deep sequencing, respectively. The results demonstrated that efSaCas9 possesses higher cleavage activity and fidelity than SaCas9-HF at the most endogenous sites in human cells. Collectively, our study provides insights for the rational selection of suitable SaCas9 for human genome editing.
CRISPR/Cas9（规律成簇间隔短回文重复/CRISPR 相关内切酶 Cas9）核酸酶已被广泛应用于基因组工程。金黄色葡萄球菌 Cas9（SaCas9）结构紧凑，可被包装在 AAV（腺相关病毒）载体中进行体内基因编辑。然而，野生型 SaCas9 会诱导不必要的脱靶突变，极大地限制了其应用。迄今为止，已有两种报道的具有高保真度的 SaCas9 变体，包括我们之前研究中的 efSaCas9 和 SaCas9-HF。然而，哪种变体具有更好的保真度和更高的活性仍不清楚。在这里，我们通过荧光报告系统和靶深测序分别在人细胞中对 efSaCas9 和 SaCas9-HF 进行了平行比较。结果表明，efSaCas9 在人细胞中大多数内源性靶位的切割活性和保真度均高于 SaCas9-HF。总之，我们的研究为合理选择适合人类基因组编辑的 SaCas9 提供了思路。
