Precision Genome Editing Using Bacteriophage-derived Peptides

Protein and small molecule-based CRISPR inhibitors have been developed as selective off-switches to modulate CRISPR-Cas activity. These anti-CRISPR agents have important therapeutic implications because of their capability to improve the specificity of CRISPR-Cas in human cells. In this study, we reported the discovery of Cas9-inactivating peptides from inoviridae bacteriophages. These peptides, derived from the periplasmic domain of phage major coat protein G8P (G8PPD), can inhibit the in vitro cleavage activity of purified Streptococcus pyogenes Cas9 (SpyCas9) proteins in an allosteric manner. Ectopic expression of full-length G8P (G8PFL) or G8PPD in human cells can inactivate the genome-editing activity of SpyCas9 with minimum alterations of the mutation patterns. Most importantly, timed delivery of G8PPD can reduce the off-target activity of co-transfected SpyCas9 without compromising its on-target activity. Therefore, the G8Ps discovered in the current study represent the first anti-CRISPR peptides that can improve the specificity of constitutively expressed SpyCas9, implying the potential applications of G8Ps for precision genome editing.