CRISPR-Cas systems are transformative tools with real-world applications from genetic engineering to gene drives. Critical to the effective use of CRISPR biotechnology is both the discovery and development of anti-CRISPRs - phage derived proteins capable of potently inhibiting CRISPR functionality. However, the discovery of naturally occurring anti-CRISPRs can be challenging and many biotechnologically relevant Cas effectors have no inhibitors to tune or regulate their activity. Here, we present an approach that leverages de novo protein design and advanced in silico methods, such as RF Diffusion, to create new-to-nature proteins that control CRISPR-Cas activity. We demonstrate that these designs, referred to as AI-designed anti-CRISPRs (AIcrs), are capable of highly potent and specific inhibition of the target protein, CRISPR-Cas13. Comprehensive validation through biochemical assays and structural biology confirms their efficacy, and we demonstrate AIcr utility in controlling the anti-phage activity of CRISPR-Cas13 in bacteria.
The ability to rapidly design custom inhibitors of CRISPR-Cas machinery will contribute to the ongoing development of gene editors for application in the clinic and safeguard against their misuse. This work also underscores the revolution in protein design and highlights the urgent need to leverage these advancements for future biotechnological applications.