Lin Ping, Qin Shugang, Pu Qinqin, Wang Zhihan, Wu Qun, Gao Pan, Schettler Jacob, Guo Kai, Li Rongpeng, Li Guoping, Huang Canhua, Wei Yuquan, Gao George Fu, Jiang Jianxin, Wu Min
Department of Biomedical Sciences, School of Medicine and Health Sciences, University of North Dakota, Grand Forks, ND 58203, USA; Wound Trauma Medical Center, State Key Laboratory of Trauma, Burns and Combined Injury, Daping Hospital, Army Medical University, Chongqing 400042, China.
Department of Biomedical Sciences, School of Medicine and Health Sciences, University of North Dakota, Grand Forks, ND 58203, USA; State Key Laboratory of Biotherapy, West China Hospital, Sichuan University, Chengdu, Sichuan 610041, China.
Mol Cell. 2020 Jun 4;78(5):850-861.e5. doi: 10.1016/j.molcel.2020.03.033. Epub 2020 Apr 28.
Cas13 has demonstrated unique and broad utility in RNA editing, nucleic acid detection, and disease diagnosis; however, a constantly active Cas enzyme may induce unwanted effects. Bacteriophage- or prophage-region-encoded anti-CRISPR (acr) gene molecules provide the potential to control targeting specificity and potency to allow for optimal RNA editing and nucleic acid detection by spatiotemporally modulating endonuclease activities. Using integrated approaches to screen acrVI candidates and evaluate their effects on Cas13 function, we discovered a series of acrVIA1-7 genes that block the activities of Cas13a. These VI-A CRISPR inhibitors substantially attenuate RNA targeting and editing by Cas13a in human cells. Strikingly, type VI-A anti-CRISPRs (AcrVIAs) also significantly muffle the single-nucleic-acid editing ability of the dCas13a RNA-editing system. Mechanistically, AcrVIA1, -4, -5, and -6 bind LwaCas13a, while AcrVIA2 and -3 can only bind the LwaCas13-crRNA (CRISPR RNA) complex. These identified acr molecules may enable precise RNA editing in Cas13-based application and study of phage-bacterium interaction.
Cas13在RNA编辑、核酸检测和疾病诊断中已展现出独特且广泛的用途；然而，持续活跃的Cas酶可能会引发不良影响。噬菌体或原噬菌体区域编码的抗CRISPR（acr）基因分子为控制靶向特异性和效力提供了潜力，从而通过时空调节核酸内切酶活性实现最佳的RNA编辑和核酸检测。通过综合方法筛选acrVI候选物并评估它们对Cas13功能的影响，我们发现了一系列可阻断Cas13a活性的acrVIA1 - 7基因。这些VI - A类CRISPR抑制剂在人类细胞中显著减弱了Cas13a对RNA的靶向和编辑作用。令人惊讶的是，VI - A类抗CRISPR蛋白（AcrVIAs）也显著抑制了dCas13a RNA编辑系统的单核酸编辑能力。从机制上讲，AcrVIA1、-4、-5和-6与LwaCas13a结合，而AcrVIA2和-3仅能与LwaCas13 - crRNA（CRISPR RNA）复合物结合。这些已鉴定的acr分子可能有助于在基于Cas13的应用以及噬菌体 - 细菌相互作用研究中实现精确的RNA编辑。
