Systematic discovery of natural CRISPR-Cas12a inhibitors

Cas12 inhibitors join the anti-CRISPR family Bacteria and their phages continually coevolve in a molecular arms race. For example, phages use anti-CRISPR proteins to inhibit the bacterial type I and II CRISPR systems (see the Perspective by Koonin and Makarova). Watters et al. and Marino et al. used bioinformatic and experimental approaches to identify inhibitors of type V CRISPR-Cas12a. Cas12a has been successfully engineered for gene editing and nucleic acid detection. Some of the anti-Cas12a proteins identified in these studies had broad-spectrum inhibitory effects on Cas12a orthologs and could block Cas12a-mediated genome editing in human cells. Science, this issue p. 236, p. 240; see also p. 156 CRISPR-Cas12a inhibitors that block gene editing in human cells are identified. Cas12a (Cpf1) is a CRISPR-associated nuclease with broad utility for synthetic genome engineering, agricultural genomics, and biomedical applications. Although bacteria harboring CRISPR-Cas9 or CRISPR-Cas3 adaptive immune systems sometimes acquire mobile genetic elements encoding anti-CRISPR proteins that inhibit Cas9, Cas3, or the DNA-binding Cascade complex, no such inhibitors have been found for CRISPR-Cas12a. Here we use a comprehensive bioinformatic and experimental screening approach to identify three different inhibitors that block or diminish CRISPR-Cas12a–mediated genome editing in human cells. We also find a widespread connection between CRISPR self-targeting and inhibitor prevalence in prokaryotic genomes, suggesting a straightforward path to the discovery of many more anti-CRISPRs from the microbial world.