CRISPR-Cas13 Inhibitors Block RNA Editing in Bacteria and Mammalian Cells
Authors: Qun Wu
Kai Guo
Guoping Li
Qinqin Pu
Jianxin Jiang
Shugang Qin
Rongpeng Li
Ping Lin
Min Wu
Canhua Huang
Jacob Schettler
Yuquan Wei
Pan Gao
Zhihan Wang
George Fu Gao
Journal: Molecular Cell (2020)
Discipline: MOLECULAR BIOLOGY & GENETICS
Keywords: AcrVIA; Cas13a; RNA editing; RNA targeting; anti-CRISPR.
Affiliations: 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., Department of Biomedical Sciences, School of Medicine and Health Sciences, University of North Dakota, Grand Forks, ND 58203, USA; West China School of Basic Medical Sciences and Forensic Medicine, Sichuan University, Chengdu, Sichuan 610041, China., Department of Biomedical Sciences, School of Medicine and Health Sciences, University of North Dakota, Grand Forks, ND 58203, USA; Department of Pediatrics, Ruijin Hospital affiliated to Shanghai Jiao Tong University of Medicine, Shanghai 200025, China., Department of Biomedical Sciences, School of Medicine and Health Sciences, University of North Dakota, Grand Forks, ND 58203, USA., Key Laboratory of Biotechnology for Medicinal Plants of Jiangsu Province, Jiangsu Normal University, Xuzhou, Jiangsu 221116, China., Inflammations & Allergic Diseases Research Laboratory, Affiliated Hospital of Southwest Medical University, Luzhou, Sichuan 646004, China., West China School of Basic Medical Sciences and Forensic Medicine, Sichuan University, Chengdu, Sichuan 610041, China., State Key Laboratory of Biotherapy, West China Hospital, Sichuan University, Chengdu, Sichuan 610041, China., Chinese Academy of Sciences (CAS) Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China; Savaid Medical School, University of Chinese Academy of Sciences, Beijing 101408, China; National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 102206, China; Research Network of Immunity and Health, Beijing Institutes of Life Science, Chinese Academy of Sciences, Beijing 100101, China., Wound Trauma Medical Center, State Key Laboratory of Trauma, Burns and Combined Injury, Daping Hospital, Army Medical University, Chongqing 400042, China. Electronic address: ., Department of Biomedical Sciences, School of Medicine and Health Sciences, University of North Dakota, Grand Forks, ND 58203, USA. Electronic address: .

Abstract:  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. 

Retracted: No

PubPeer comments:
 Readers should become aware of a recent preprint entitled Lack of Cas13a inhibition by anti-CRISPR proteins from Leptotrichia prophages that calls into question part of the conclusions obtained in Lin et al., 2020 study. https: www.biorxiv.org content 10.1101 2021.05.27.445852v1 From the abstract: CRISPR systems are prokaryotic adaptive immune systems that use RNA-guided Cas nucleases to recognize and destroy foreign genetic elements, like bacteriophages and plasmids. To overcome CRISPR immunity, phages have evolved diverse families of anti-CRISPR proteins (Acrs), each of which inhibits the nucleic acid binding or cleavage activities of specific Cas protein families. **Recently, Lin et al. (2020) described the discovery and characterization of 7 different Acr families (AcrVIA1-7) that inhibit type VI-A CRISPR systems, which use the nuclease Cas13a to perform RNA-guided RNA cleavage. In this Matters Arising article, we detail several inconsistencies that question the results reported in the Lin et al. (2020) study. These include inaccurate bioinformatics analyses, as well as reported experiments involving bacterial strains that are impossible to construct. The authors were unable to provide their published strains with which we might reproduce their experiments. We independently tested the Acr sequences described in Lin et al. (2020) in two different Cas13 inhibition assays, but could not detect anti-CRISPR activity. Taken together, our data and analyses prompt us to question the claim that AcrVIA1-7 reported in Lin et al. are bona fide type VI anti-CRISPR proteins.** , 
 #1 I am the first author, who performed the assays and analyzed data. I highly appreciate the comments and questions raised in this preprint, which are extremely helpful for us to find any deficiency and validate our results. We will examine the original data, bioinformatic analyses, potential technical issues, and will perform necessary analyses and respond as soon as possible. Thank you again. Sincerely yours, Ping Lin , 
 Johnson et al. independently assayed the activity of 7 type VI-A anti-CRISPRs (Acrs) published by Lin et al. (2020) but found no Cas13a inhibition. The authors detail inconsistencies with the constructed strains, plasmids, and bioinformatics analyses by Lin et al. https: www.cell.com molecular-cell fulltext S1097-2765(22)00436-1 ,