Integrated approaches discovered multiple novel anti-Cas13 molecules to enhance RNA editing capacity

Abstract

Abstract We have used an integrated approach to finding anti-CRISPR (Acr) molecules (acrVIA1-7) to repress type VI-A CRISPR (Cas13a, an RNA targeting Cas) system. This involves a combination of transcription-translation assay (TXTL), self-targeting screen system (STSS) and guilt-by-association. We started with bioinformatics-based STSS screening technique to discover strains with self-targeting spacers by Cas13 in diverse strains containing type VI CRISPR-Cas systems, identifying the Leptotrichia wadei F0279 strain having self-targeting protospacers. Then we utilized a guilt-by-association method, a total of 11 potential Acrs candidates adjacent to aca genes have been characterized. Next, a TXTL system was applied to functionally evaluate the anti-CRISPR activities of these putative Acrs, leading to the discovery of five inhibitors of type VI-A CRISPR-Cas13a systems (AcrVIA1-5). Finally, we used second iterative method based on the known acr genes (acrIC1, acrIIA1 and acrIIC4) in strains possessing self-targets, resulting in identification of Cas13a inhibitors AcrVIA6-7, as well as AcrIB1 that inhibits type I-B CRISPR-Cas systems. These newly discovered Acrs exhibited the activities of inhibiting relevant Cas proteins in bacteria and mammalian cells, such as HEK293 cells. Hence, these newly identified acr molecules may have broad usages, such as improvement of RNA editing in Cas13-based application for human disease intervention, application in biotechnology, and study of phage-bacterium interaction.