Allosteric inhibition of CRISPR-Cas9 by bacteriophage-derived peptides
Cui, Yan-ru1,2,3,4; Wang, Shao-jie1; Chen, Jun5; Li, Jie1,2,3,4; Chen, Wenzhang1; Wang, Shuyue1,2,3,4; Meng, Bing1; Zhu, Wei1; Zhang, Zhuhong6; Yang, Bei1; Jiang, Biao1; Yang, Guang1; Ma, Peixiang1; Liu, Jia1
Source PublicationGENOME BIOLOGY
AbstractBackground 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. Results 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. Conclusion 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.
KeywordCRISPR-Cas9 Inoviridae bacteriophage Major coat protein G8P Allosteric inhibition Off-target activity
Indexed BySCI
Funding ProjectShanghaiTech University Startup Fund[2019F0301-000-01]
WOS Research AreaBiotechnology & Applied Microbiology ; Genetics & Heredity
WOS SubjectBiotechnology & Applied Microbiology ; Genetics & Heredity
WOS IDWOS:000517486500001
Original Document TypeArticle
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Document Type期刊论文
Corresponding AuthorMa, Peixiang; Liu, Jia
Affiliation1.ShanghaiTech Univ, Shanghai Inst Adv Immunochem Studies, Shanghai 201210, Peoples R China
2.ShanghaiTech Univ, Sch Life Sci & Technol, Shanghai, Peoples R China
3.Chinese Acad Sci, Shanghai Inst Biochem & Cell Biol, CAS Ctr Excellence Mol Cell Sci, Shanghai, Peoples R China
4.Univ Chinese Acad Sci, Beijing, Peoples R China
5.Zhejiang Univ, Coll Life Sci, Hangzhou 310058, Zhejiang, Peoples R China
6.Yantai Univ, Key Lab Mol Pharmacol & Drug Evaluat Yantai Univ, Collaborat Innovat Ctr Adv Drug Delivery Syst & B, Sch Pharm,Minist Educ, Yantai 264005, Shandong, Peoples R China
First Author AffilicationShanghai Institute for Advanced Immunochemical Studies;  School of Life Science and Technology
Corresponding Author AffilicationShanghai Institute for Advanced Immunochemical Studies
First Signature AffilicationShanghai Institute for Advanced Immunochemical Studies
Recommended Citation
GB/T 7714
Cui, Yan-ru,Wang, Shao-jie,Chen, Jun,et al. Allosteric inhibition of CRISPR-Cas9 by bacteriophage-derived peptides[J]. GENOME BIOLOGY,2020,21(1).
APA Cui, Yan-ru.,Wang, Shao-jie.,Chen, Jun.,Li, Jie.,Chen, Wenzhang.,...&Liu, Jia.(2020).Allosteric inhibition of CRISPR-Cas9 by bacteriophage-derived peptides.GENOME BIOLOGY,21(1).
MLA Cui, Yan-ru,et al."Allosteric inhibition of CRISPR-Cas9 by bacteriophage-derived peptides".GENOME BIOLOGY 21.1(2020).
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