Engineered FnCas12a with enhanced activity through directional evolution in human cells.

• 发表时间: Jan-Jun 2021
• 来源期刊: J Biol Chem
• DOI: 10.1016/j.jbc.2021.100394
• PMID: 33567342
• 文章类型: Journal Article
• 中科院分区: Q2
• 方向: 生物
• 作者列表: Liu X,Liu X,Zhou C,Lv J,He X,Liu Y,Xie H,Wang B,Lv X,Tang L,Li M,Liu C,Zhao J,Liu Y,Song Z,Gu F

关键词: FnCas12a; PAM; RS1; activity; variant

Mesh: Bacterial Proteins / genetics metabolism; CRISPR-Associated Proteins / genetics metabolism; CRISPR-Cas Systems; Cells, Cultured; Endodeoxyribonucleases / genetics metabolism; Evolution, Molecular; Eye Proteins / genetics; Francisella / enzymology genetics isolation & purification; Gene Editing / methods; Humans; Mutation; Protein Engineering / methods; Retinoschisis / genetics metabolism pathology; Selection, Genetic; Structure-Activity Relationship

Abstract

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.