Allosteric inhibition of CRISPR-Cas9 by bacteriophage-derived peptides
Allosteric inhibition of CRISPR-Cas9 by bacteriophage-derived peptides
复制标题
噬菌体衍生肽对 CRISPR-Cas9 的变构抑制。
DOI：
10.1186/s13059-020-01956-x
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发表时间：
2020-02-26
期刊：
影响因子：
12.3
通讯作者：
Liu, Jia
中科院分区：
文献类型：
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作者：
Cui, Yan-ru;Wang, Shao-jie;Liu, Jia
CRISPR-Cas9已被开发为各种传染病和遗传病的治疗剂。在许多临床相关应用中，组成型活性CRISPR-Cas9被递送到人类细胞中，而没有时间控制系统。CRISPR-Cas9的过度和延长的表达可导致升高的脱靶切割。随着时间和剂量调节CRISPR-Cas9活性的需要产生了开发CRISPR-Cas关闭开关的需求。基于蛋白质和小分子的CRISPR-Cas抑制剂已在先前的研究中报道。结果我们报道了从inoviridae噬菌体中发现的Cas9抑制肽。这些肽来源于噬菌体主要外壳蛋白G8 P（G8 P（PD））的周质结构域，可以以变构方式抑制化脓性链球菌Cas9（SpCas 9）蛋白的体外活性。重要的是，G8 P（PD）对SpCas 9的抑制活性取决于向导RNA添加的顺序。在人类细胞中异位表达全长G8 P（G8 P（FL））或G8 P（PD）可以抑制SpyCas 9的基因组编辑活性，而突变模式的改变最小。此外，与完全消除CRISPR-Cas9的细胞活性的抗CRISPR蛋白AcrII 4A不同，G8 P共转染可以降低共转染的SpCas 9的脱靶活性，同时保留其中靶活性。结论本研究发现的G8 Ps代表了第一个可以变构抑制CRISPR-Cas9的抗CRISPR肽。这一发现可能为开发用于精确基因组工程的下一代CRISPR-Cas抑制剂提供见解。
Background 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.