State Key Laboratory and Key Laboratory of Vision Science, Ministry of Health and Zhejiang Provincial Key Laboratory of Ophthalmology and Optometry, School of Ophthalmology and Optometry, Eye Hospital, Wenzhou Medical University, Wenzhou, Zhejiang, China.
The Second Clinical College, The Second Affiliated Hospital and Yuying Children's Hospital of Wenzhou Medical University, Wenzhou, Zhejiang, China.
J Biol Chem. 2021 Jan-Jun;296:100394. doi: 10.1016/j.jbc.2021.100394. Epub 2021 Feb 7.
Clustered regularly interspaced short palindromic repeat-Cas12a has been harnessed to manipulate the human genome; however, low cleavage efficiency and stringent protospacer adjacent motif hinder the use of Cas12a-based therapy and applications. Here, we have described a directional evolving and screening system in human cells to identify novel FnCas12a variants with high activity. By using this system, we identified IV-79 (enhanced activity FnCas12a, eaFnCas12a), which possessed higher DNA cleavage activity than WT FnCas12a. Furthermore, to widen the target selection spectrum, eaFnCas12a was engineered through site-directed mutagenesis. eaFnCas12a and one engineered variant (eaFnCas12a-RR), used for correcting human RS1 mutation responsible for X-linked retinoschisis, had a 3.28- to 4.04-fold improved activity compared with WT. Collectively, eaFnCas12a and its engineered variants can be used for genome-editing applications that requires high activity.
成簇规律间隔短回文重复-Cas12a 已被用于操纵人类基因组；然而，低切割效率和严格的邻近基序限制了 Cas12a 为基础的治疗和应用。在这里，我们在人类细胞中描述了一个定向进化和筛选系统，以鉴定具有高活性的新型 FnCas12a 变体。通过使用这个系统，我们鉴定了 IV-79（增强活性 FnCas12a，eaFnCas12a），它具有比 WT FnCas12a 更高的 DNA 切割活性。此外，为了拓宽靶标选择谱，通过定点突变工程改造了 eaFnCas12a。eaFnCas12a 和一个工程化变体（eaFnCas12a-RR），用于纠正导致 X 连锁性视网膜劈裂的人类 RS1 突变，与 WT 相比，活性提高了 3.28 至 4.04 倍。总之，eaFnCas12a 和其工程化变体可用于需要高活性的基因组编辑应用。
