Molecular basis for DNA cleavage by the hypercompact Cas12j-SF05
Zhiqiang Duan, Xi Zhang, Juntao Zhang, Shanshan Li, Ruiheng Liu, Jie Sun, Zhao Qingzhi, Nannan Jia, Ning Jia, Jian‐Kang Zhu, Ning Jia, Jian‐Kang Zhu
Abstract
The CRISPR-Cas systems provide bacteria and archaea with adaptive immunity against foreign invading viruses and plasmids 1 . Based on their Cas gene composition, CRISPR-Cas systems are categorized into two classes with six types: Class 1 (type I, III, and IV) and Class 2 (type II, V, and VI) 1 . The compact and portable single-protein effectors from Class 2 systems, such as type II SpyCas9 (1368 aa) 2 or type V Cas12a (~1300 aa) 3 , are widely used in genome editing, gene regulation, and genome imaging across various organisms. However, their large gene sizes pose a challenge for packaging into viral vectors, which can hinder their delivery and thus limit their utility in genome engineering applications. Cas12f family proteins represent the most compact Class 2 CRIPSR effectors (400–700 aa) reported to date and are capable of editing genes in both bacteria and human cells 3 . Distinct from Cas12f proteins that rely on the presence of both crRNA and tracrRNA and cleave target dsDNA in a dimerization-dependent manner 4 , 5 , the recently identified CasΦ (Cas12j) family proteins derived from huge bacteriophages rely on crRNA only and cleave target dsDNA in a monomeric form 6 . The Cas12j proteins also represent a minimal functional CRISPR-Cas system (700–800 aa), which can effectively cleave target dsDNA in vitro and mediate gene editing in human and plant cells 6 , 7 , 8 , 9 . Thus, Cas12j family members hold great potential for genome editing and diagnostics. While the reported Cas12j proteins exhibited efficient editing of endogenous genes in mammalian cells 10 , they displayed limited gene editing activity in plants, with gene editing efficiencies of ~0.3% and ~6% mediated by wild-type and engineered CasΦ-2 (Cas12j-2) variants, respectively, observed in the dicot plant Arabidopsis . In monocot plants, the reported heritable gene editing efficiency of Cas12j proteins is further limited, with only up to 1.2% in stably transformed rice lines 9 .