De novodesign of potent CRISPR-Cas13 inhibitors

CRISPR-Cas systems are transformative tools for gene editing which can be tuned or controlled by anti-CRISPRs (Acrs) - phage derived inhibitors that regulate CRISPR-Cas activity. However, Acrs that are capable of inhibiting biotechnologically relevant CRISPR systems are relatively rare and challenging to discover. To overcome this limitation, we describe a highly successful, rapid, and generalisable approach that leveragesde novoprotein design to develop new-to-nature proteins for controlling CRISPR-Cas activity. Using CRISPR-Cas13 as a representative example, we demonstrate that AI-designed anti-CRISPRs (AIcrs) are capable of highly potent and specific inhibition of CRISPR-Cas13 proteins. We present a comprehensive workflow for design validation and demonstrate AIcrs functionality in controlling CRISPR-Cas13 activity in bacteria. The ability to design bespoke inhibitors of Cas effectors will contribute to the ongoing development of CRISPR-Cas tools in diverse applications across research, medicine, agriculture, and microbiology.