Highly stable Cas9 promotes HBV genome destruction by antagonizing HSC70-mediated degradation

As a naturally existing adaptive immune system of prokaryotes against phages and foreign genetic materials, the CRISPR/Cas9 system has been widely used to combat various viral infections. However, its ability to destroy the constantly replicating viral genome and subsequently clear viral infections still needs further improvement. This study found that Cas9 protein was mainly degraded through the chaperone-mediated autophagy (CMA)-lysosome pathway in human cells, which was mediated by the binding between heat shock cognate protein 70 (HSC70) and Cas9 protein. HRS could stabilize Cas9 protein by competing with HSC70 to bind to Cas9 and subsequently inhibiting its degradation via the CMA-lysosome pathway. The stability of Cas9 protein with mutant KFERQ-like motifs located at aa 670-674 and aa 894-898 was significantly increased by antagonizing the HSC70-mediated CMA degradation, thus this Cas9 mutant was referred to as a highly stable Cas9 (HSCas9). The enhanced ability of HSCas9 to destroy the constantly replicating hepatitis B virus (HBV) genome promoted the CRISPR/Cas9 system to clear HBV infection without exhibiting cytotoxicity or increasing off-target effects. In summary, this study uncovers the degradation mechanism of Cas9 protein in human cells and provides a strategy to enhance the ability of the CRISPR/Cas9 system to clear HBV infection.Abbreviations: ALP: autophagy-lysosome pathways; AR7: 7-Chloro-3-(4-methylphenyl)-2H-1,4-benzoxazine; cccDNA: covalently closed circular DNA; CMA: chaperone-mediated autophagy; CRISPR/Cas9: clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated nuclease 9 (Cas9); gRNA: single-guide RNA; HBV: hepatitis B virus; HRS: hepatocyte growth factor-regulated tyrosine kinase substrate; HSC70: heat shock cognate protein 70; HSCas9: highly stable Cas9; rcDNA: relaxed circular DNA; SNP: single nucleotide polymorphism; UPS: ubiquitin-proteasome system.