Allosteric inhibition of CRISPR-Cas9 by bacteriophage-derived peptides

CRISPR-Cas9 has been developed as a therapeutic agent for various infectious and genetic diseases. In many clinically relevant applications, constitutively active CRISPR-Cas9 is delivered into human cells without a temporal control system. Excessive and prolonged expression of CRISPR-Cas9 can lead to elevated off-target cleavage. The need for modulating CRISPR-Cas9 activity over time and dose has created the demand of developing CRISPR-Cas off switches. Protein and small molecule-based CRISPR-Cas inhibitors have been reported in previous studies.

We report the discovery of Cas9-inhibiting peptides from inoviridae bacteriophages. These peptides, derived from the periplasmic domain of phage major coat protein G8P (G8P_PD), can inhibit the in vitro activity of Streptococcus pyogenes Cas9 (SpCas9) proteins in an allosteric manner. Importantly, the inhibitory activity of G8P_PD on SpCas9 is dependent on the order of guide RNA addition. Ectopic expression of full-length G8P (G8P_FL) or G8P_PD in human cells can inactivate the genome-editing activity of SpyCas9 with minimum alterations of the mutation patterns. Furthermore, unlike the anti-CRISPR protein AcrII4A that completely abolishes the cellular activity of CRISPR-Cas9, G8P co-transfection can reduce the off-target activity of co-transfected SpCas9 while retaining its on-target activity.

G8Ps discovered in the current study represent the first anti-CRISPR peptides that can allosterically inactivate CRISPR-Cas9. This finding may provide insights into developing next-generation CRISPR-Cas inhibitors for precision genome engineering.