Allosteric inhibition of CRISPR-Cas9 by bacteriophage-derived peptides

Yan-Ru Cui,Jun Chen,Wenzhang Chen,Jie Li,Shao-Jie Wang,Zhuhong Zhang,Biao Jiang,Jia Liu,Bing Meng,Shuyue Wang,Wei Zhu,Bei Yang,Peixiang Ma,Guang Yang

doi:10.1186/s13059-020-01956-x

Abstract

BackgroundCRISPR-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.ResultsWe report the discovery of Cas9-inhibiting peptides from inoviridae bacteriophages. These peptides, derived from the periplasmic domain of phage major coat protein G8P (G8PPD), can inhibit the in vitro activity of Streptococcus pyogenes Cas9 (SpCas9) proteins in an allosteric manner. Importantly, the inhibitory activity of G8PPD on SpCas9 is dependent on the order of guide RNA addition. 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. 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.ConclusionG8Ps 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.

Highlights

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9 has been developed as a therapeutic agent for various infectious and genetic diseases
Intact M13 phage inhibits the in vitro DNA cleavage activity of SpCas9 In a conventional phage display experiment, we surprisingly discovered that intact M13 phage [51] itself could block the DNA cleavage activity of purified SpCas9 proteins in a dose-dependent manner with an approximate half maximum inhibitory concentration (IC50) of 5 nM (Fig. 1a), which corresponds to a phage titer of 3 × 109 PFU/μL
The order-of-addition-dependent inhibition suggested that competition for sgRNA-binding site in SpCas9 is a possible mechanism of the inhibitory activity of M13 phage

Summary

Introduction

CRISPR-Cas has been developed as a therapeutic agent for various infectious and genetic diseases. In many clinically relevant applications, constitutively active CRISPR-Cas is delivered into human cells without a temporal control system. Excessive and prolonged expression of CRISPR-Cas can lead to elevated off-target cleavage. Protein and small molecule-based CRISPR-Cas inhibitors have been reported in previous studies. Invader DNA is captured and integrated into bacterial genome as CRISPR array. Type II CRISPR-Cas systems function with streamlined components comprising of a single nuclease protein such as Cas9 [4]. The modular and programmable features make CRISPR-Cas one of the most widely used tools for genome engineering applications [5,6,7,8].

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Discussion

Conclusion