Stable Cas9 helps destroy hepatitis B virus DNA by blocking its breakdown through a cellular heat shock protein

HuH7, HepG2, and HeLa cells were maintained in a Dulbecco's modified Eagle medium (DMEM) (Corning, New York, USA) supplemented with 10% fetal bovine serum (FBS) (PAN, Adenbach, Bavaria, Germany) and 1% penicillin/streptomycin (PS), in a 5% CO₂ incubator at 37℃. The stable HBV-expressing cell line HepAD38 was kindly provided by Prof. Ningshao Xia at Xiamen University, and was cultured with DMEM containing 10% FBS, 400 μg/mL G418 (Amresco, Solon, Washington, USA), and 4 μg/mL Doxycycline (Merck, Kenilworth, New Jersey, USA). HepG2-NTCP cells were kindly provided by Prof. Kuanhui Xiang at Peking University and were cultured with DMEM containing 10% FBS and 400 μg/mL G418. LAMP2A gene knockout (KO) HEK293 T cells, SQSTM1/p62 gene KO HEK293 T cells, ATG3 gene KO HEK293 T cells, ATG5 gene KO HEK293 T cells, and the corresponding control cells were constructed [29] and kindly provided by Prof. Hongxin Huang at Sun Yat-sen University, and were cultured with DMEM containing 10% FBS, 1% PS, and 1 μg/mL puromycin (Thermo Fisher Scientific, Waltham, Massachusetts, USA). HeLa-Cas9-Flag cells stably expressing Cas9 [30], HepG2-NTCP-Cas9 cells stably expressing Cas9 and HepG2-NTCP-HSCas9 cells expressing HSCas9 were constructed in our laboratory and were cultured with DMEM containing 10% FBS, 1% PS, and 1 μg/mL puromycin (Thermo Fisher Scientific, Waltham, Massachusetts, USA).

Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) was used to transfect the plasmid, and Lipofectamine RNAiMAX (Thermo Fisher Scientific, Waltham, MA, USA) was used to transfect siRNA oligonucleotide duplex (RiBobio, Guangzhou, Guangdong, China), according to the manufacturer's protocol. MG132 (Selleck, Houston, TX, USA), HCQ (Selleck, Houston, TX, USA), BafA1 (MCE, New Jersey, USA), Cycloheximide (MCE, New Jersey, USA), AR7 (MCE, New Jersey, USA), and LY294002 (MCE, New Jersey, USA) were used to treat cells, respectively.

The plasmid pBB4.5-HBV1.3 was previously constructed in our laboratory. The PCDH-HSC70-HA plasmid was constructed by amplifying the HSC70 coding sequence attached with an HA tag and inserting it into the PCDH plasmid digested with BamHI and XbaI. Based on the pSpCas9 (BB)-2A-EGFP (PX458) plasmid (Addgene, Cambridge, MA), the PX458-Cas9M1, PX458-Cas9M2, PX458-Cas9M3, PX458-Cas9M4, and PX458-HSCas9 plasmids were constructed using site-directed mutagenesis using the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA). Based on the lentiCRISPRv2 plasmid (Addgene, Cambridge, MA), the lenti-HSCas9 plasmid was constructed by site-directed mutagenesis. The primer sequences used for plasmid construction are shown in Supplementary Table S1.

Reverse transcription was performed using the Transcriptor First Strand cDNA Synthesis Kit (Roche, Basel, Kanton Basel, Switzerland). The mRNA levels of Cas9, LAMP2A, and ACTB were detected by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) (SYBR Green method) using an Applied Biosystems Step One Plus Real-Time PCR system (Thermo Fisher Scientific, Waltham, Massachusetts, USA). The primers used to detect the levels of gRNA and mRNA are listed in Supplementary Table S2.

The total DNA of cells was extracted using QIAamp DNA Mini Kit (Qiagen, Hilden, Germany), according to the manufacturer's instructions. The on-target cleavage efficiency of the HBV genome was assessed by PCR using the primers outside of the two cleavage sites of gHBV1/Cas9 and gHBV2/Cas9, HBV-F (5′-GACTCGTGGTGGACTTCTCTCAA-3′), and HBV-R (5′-CTGACTACTAATTCCCTGGATGCT-3′). The PCR products were detected by 1.5% agarose gel electrophoresis and purified for sequencing by NucleoSpin Gel and PCR Clean-up Kit (Macherey-Nagel). The purified PCR products were sequenced by Sangon Biotech (Shanghai, China).

Further, the on-target cleavage efficiency of the HBV genome was assessed by the next-generation sequencing. Specifically, the HBV genome was amplified with the primers outside of the two cleavage sites of gHBV1/Cas9 and gHBV2/Cas9, HBV-F1 (5′-ATCCTGCCTTGATGCCTTTA-3′), and HBV-R1 (5′-ACAGAGCTGAGGCGGTGTCG-3′). The next-generation sequencing of PCR products was performed by Sangon Biotech (Shanghai, China), and 150 bp paired-end reads were generated. The raw reads were filtered by Trimmomatic (Version 0.39) to obtain clean reads. Clean reads were aligned to the HBV reference genome using a Burrows–Wheeler Aligner (BWA, Version 0.7.17). After alignment, SAMtools (Version 1.9) was used to convert the alignment result format, and GATK (Version 4.1.1.0) was used to call out all variants, including insertion or deletion (indel) variants. The on-target cleavage efficiency of the HBV genome was the number of indel variant reads at the cleavage site divided by the total number of reads at the cleavage site.

The nuclear DNA was extracted as the protocol previously reported [31]. Briefly, cells were lysed with TE buffer and 10% SDS. The cell lysates were added with 5 M NaCl and were precipitated for at least 16 h at 4°C, and then the supernatant was collected after centrifugation. Finally, the nuclear DNA was purified by phenol/chloroform and ethanol. The level of the complete HBV cccDNA was detected by qPCR using the primers between the two cleavage sites of gHBV1/Cas9 and gHBV2/Cas9, cccDNA-F: GGGGCGCACCTCTCTTTA, and cccDNA-R: AGGCACAGCTTGGAGGC.

The levels of HBsAg and HBeAg were detected by chemiluminescence immunoassay kits (Autobio Diagnostics Co., Zhengzhou, China), according to the manufacturer's instructions.

Co-IP assay and Western blotting were performed as we previously described [32]. The protein levels were detected by the chemiluminescence imaging analysis system (Tanon, Shanghai, China) or the Odyssey infrared imaging system (LICOR, Lincoln, Nebraska, USA). The corresponding antibodies are shown in Supplementary Table S3.

Cells were cultured on the Petri dish for confocal (D35-20-1-N, Cellvis), and then fixed with 4% PFA and permeabilized with 0.1% TritonX-100 or Tween-20. Subsequently, the cells were incubated with the primary antibodies at 4°C overnight. After washing three times with 1 × PBS, the cells were incubated with the secondary antibodies for 1 h at room temperature, and then were observed under a STELLARIS 8 confocal laser scanning microscope (Leica, Frankfurt, Hesse-Darmstadt, Germany). The corresponding antibodies are shown in Supplementary Table S3.

A total of 1 × 10⁴ cells were seeded in 96-well plates with six duplicates, and then the cell viability was detected using a Cell Counting Kit-8 (CCK-8) (Dojindo, Japan). The absorbance was measured at the wavelength of 450 nm by a SpectraMax Absorbance Reader (Molecular Devices, California, USA).

The off-target efficiency of Cas9 was assessed by the next-generation sequencing after PCR. Specifically, the off-target sites of gHBV1 in the human genome (GRCh38/hg38) were predicted by the Cas-OFFinder tool [33]. The top three predicted off-target sites were selected and listed in Supplementary Table S4. The primers used for PCR amplification are listed in Supplementary Table S5. The PCR products were sequenced by Sangon Biotech Co., Ltd. (Shanghai, China). The paired-end sequencing of the libraries was performed on the Illumina sequencing platform (NovaSeq6000, USA). Three steps filtered the raw reads: (1) Removing adaptor sequence by the Cutadapt software; (2) Removing the reads with a mean quality score <20 by PRINSEQ-lite software; (3) Removing chimaeras' sequence by Usearch software. The analyses and visualization of the off-target effects were performed by CRISPResso2. The off-target efficiency was the number of reads containing an indel variant divided by the total number of reads.

The potential off-target sites of gHBV1 and gHBV2 in the mouse genome (mm39) were also predicted by the Cas-OFFinder tool. Based on the whole-genome sequencing data, single-nucleotide polymorphism (SNP), and indel variants within 20 bp upstream and downstream of all potential off-target sites were identified and analysed.

Southern blotting was performed using the DIG High Prime DNA Labelling and Detection Starter Kit II (Roche, Basel, Kanton Basel, Switzerland). Briefly, HBV cccDNA was separated in a 1.2% agarose gel. After denaturalization and neutralization, DNA was transferred from the gel onto a Nylon membrane (Roche, Basel, Kanton Basel, Switzerland). Then the membrane was cross-linked with UV energy dosage at 120 mJ/cm² and then hybridized overnight at 42°C with a DIG-labelled HBV probe. Subsequently, the membrane was blocked and incubated with anti-digoxigenin-AP Fab fragments, followed by exposure via a ChemiScope6100 imaging device (Clinx, Shanghai, China).

HBV was collected from the supernatants of HepAD38 cells, followed by concentration and HBV DNA quantification. HepG2-NTCP-Cas9 and HepG2-NTCP-HSCas9 cells were seeded in a 6-well plate and were transfected with pU6-t-g1-t-g2-t plasmid or vector control. At 24 h post-transfection, the cells were inoculated with 500 HBV genome equivalents per cell (500 geq/cell) in the presence of 4% PEG8000 and 2% DMSO for 24 h. After HBV infection, the cells were washed with PBS 5 times, and the fresh medium with DMEM containing 2% DMSO and 2% FBS was added. Five days later, the cells and supernatants were collected for detection.

Hydrodynamic injection experiments were carried out as we previously reported [34]. Twenty-four male C57BL/6 mice (5 weeks old) were randomly divided into NC, Cas9, and HSCas9 groups, and there were 8 mice in each group. The mice in the NC group were injected with pBB4.5–1.3 × HBV, PX458, and pU6 plasmids. The mice in the Cas9 group were injected with pBB4.5–1.3 × HBV, PX458, and pU6-t-g1-t-g2-t plasmids. The mice in the HSCas9 group were injected with pBB4.5–1.3 × HBV, PX458-HSCas9, and pU6-t-g1-t-g2-t plasmids. Sera were collected at 5 and 7 days after hydrodynamic injection, and liver tissues were collected at 3 and 7 days after hydrodynamic injection, respectively.

Animal experiments were approved by the Ethics Committee of Peking University and were carried out according to the guidelines established by the Institutional Animal Care and Use Committee at Peking University Health Science Center.

Genomic DNA of three mice per group was extracted using QIAamp DNA Mini Kit (Qiagen, Hilden, Germany), according to the manufacturer's instructions. Library construction and sequencing were conducted by OE Biotech. (Shanghai, China). The raw reads were subjected to a quality check and then filtered by FASTQ (Version 0.19.5) to obtain clean reads. Clean reads were aligned to the mouse (mm39) reference genome using a Burrows–Wheeler Aligner (BWA, Version 0.7.12). After alignment, Picard (http://broadinstitute.github.io/picard/↗, Version 4.1.0.0) was employed to mark duplicate reads, and SAMtools (Version 1.4) was used to convert the alignment result format. GATK (Version 4.1.0.0) was used to call out all variants, including SNPs and indel variants. Further, the variants (SNPs and indels) shared by all samples were removed to obtain the confidence variants.

The levels of ALT and AST in the sera of mice were detected by ELISA kits (Elabscience, Wuhan, China), according to the manufacturer's instructions.

All data were presented as mean ± standard deviation (SD) of three independent experiments. Statistical analyses were performed by SPSS 24.0 software. Comparisons of the differences between groups were analysed by two-tailed Student's t-tests, and a P-value less than 0.05 was considered statistically significant.

Next, HeLa-Cas9 cells and the PX458 plasmid-transfected HEK293 T, HuH7, and HepG2 cells were treated with LY294002, a macroautophagy-specific activator [35]. As a result, there was no change in the level of Cas9 protein when the level of SQSTM1/p62 protein, a substrate protein of macroautophagy, decreased by treating with LY294002 (supplementary Figures S1E and S1F). There was also no apparent change in the level of Cas9 protein after knocking out SQSTM1/p62, ATG3, or ATG5 gene (supplementary Figures S1G–S1I). Due to the lack of a microautophagy-specific inhibitor or activator, HeLa-Cas9 cells were next treated with 7-Chloro-3-(4-methylphenyl)-2H-1,4-benzoxazine (AR7), a CMA-specific activator that promotes CMA by upregulating the transcription of the LAMP2A gene encoding a lysosomal membrane receptor–LAMP2A protein [36]. As a result, the level of Cas9 protein significantly decreased when CMA was activated by AR7 (Figures 1(C,D), and Supplementary Figures S2A–S2C). The same phenomenon was also found in HEK293 T, HuH7, and HepG2 cells (Figure 1(E) and supplementary Figures S2D and S2E). After knocking out the LAMP2A gene, the level of Cas9 protein increased (supplementary Figures S2F and S2G), and there was no apparent change in the level of Cas9 protein under AR7 treatment (supplementary Figure S2H). Furthermore, when the CMA-lysosome pathway was induced by starvation, the level of Cas9 protein also decreased with the increase of LAMP2A protein level (supplementary Figures S2I–S2K). The above results suggest that Cas9 protein is primarily degraded through the CMA-lysosome pathway.

Since most proteins degraded through the CMA pathway are mediated by molecular chaperone HSC70 and contain KFERQ-like motifs that can be bound by HSC70 protein [27], we first performed a Co-IP assay to detect the binding activity of Cas9 and HSC70 protein. The result showed that Cas9 protein could be pulled down by HSC70 (Figure 1(F)). In turn, HSC70 protein could also be pulled down by Cas9 (Figure 1(G)). Meanwhile, we found that Cas9 protein could co-localize with HSC70 and LAMP2A proteins in cells (Figure 1(H) and supplementary Figure S3A). Further, we analysed the amino acid sequence of the Cas9 protein. As a result, four KFERQ-like motifs were observed in Cas9 protein, including IRDKQ located at amino acid (aa) 670–674 in the REC3 domain, QILKE located at aa 794–798 and QRKFD located at aa 894–898 in the HNH domain, and RKDFQ located at aa 967–971 in the RuvC-III domain (Figure 1(I)). Furthermore, the level of the Cas9 protein increased when the endogenous HSC70 expression was knocked down (Figures 1(J,K), and supplementary Figures S3B–S3E). In turn, overexpressing HSC70 decreased the level of the Cas9 protein (Figure 1(L) and supplementary Figures S3F–S3H).

We and other groups have found that the Cas9 protein can be released through exosomes [30,37–39]. The biogenesis of exosomes is composed of endosomal sorting complex required for transport (ESCRT) dependent and independent pathways. ESCRT-0 component-hepatocyte growth factor-regulated tyrosine kinase substrate (HRS) usually participates in the degradation of target protein through ALP [40,41]; thus, we speculated that HRS might also be involved in the degradation of Cas9 protein. As shown in supplementary Figures S4A–S4C, the level of Cas9 protein in HeLa-Cas9 cells was significantly decreased by knocking down HRS expression, and there was no effect on the level of the Cas9 mRNA (supplementary Figure S4D). In turn, overexpressing HRS increased the level of the Cas9 protein (supplementary Figures S4E and S4F). The same phenomenon was also found in the PX458 plasmid-transfected HuH7 cells (supplementary Figures S4G–S4J). However, the level of the Cas9 protein in exosomes did not change when the release of exosomes was inhibited by knocking down TSG101 or Alix expression (supplementary Figures S4K and S4L), indicating that the Cas9 protein was mainly secreted by the ESCRT-independent pathway. There was no effect of HRS on gRNA level (supplementary Figure S4M). Further, we found that the stability of Cas9 protein was significantly decreased by knocking down HRS expression (supplementary Figures S5A and S5B). The Cas9 protein can be pulled down by HRS (supplementary Figure S5C), and the HRS protein can also be pulled down by Cas9 (supplementary Figure S5D). Furthermore, the amount of the Cas9 protein pulled down by HSC70 decreased, but the amount of the HRS protein pulled down by HSC70 increased when HRS was overexpressed (supplementary Figure S5E). Consistently, the amount of the HSC70 protein pulled down by Cas9 also decreased, but the amount of the HRS protein pulled down by Cas9 also increased when HRS was overexpressed (supplementary Figure S5F). These results suggest that HRS can stabilize Cas9 protein by competing with HSC70 to bind to Cas9 and subsequently inhibiting its degradation via CMA. These findings further confirm that the Cas9 protein is primarily degraded through the HSC70-mediated CMA-lysosome pathway.

As off-target effects are one of the major concerns for the CRISPR/Cas9 system, we next analysed whether HSCas9 increased off-target effects. After predicting the potential off-target sites of gHBV1 using the Cas-OFFinder tool [33], the next-generation sequencing was performed on the top three predicted off-target sites (supplementary Tables S4 and S5). The results showed that the off-target effects of both wild-type Cas9 and HSCas9 were very low, and HSCas9 did not increase off-target effects when compared to wild-type Cas9 (Supplementary Table S6).

Further, we explored whether the mutated KFERQ-like motifs affected the nuclease activity of the Cas9 protein. To exclude the influence of different Cas9 protein levels as much as possible, the transfection amount of wild-type Cas9, Cas9M1, Cas9M3 or HSCas9 expression plasmid was much higher than that of the 1.3×HBV plasmid. The result showed that the mutated KFERQ-like motifs of Cas9 protein did not affect the nuclease activity of Cas9 protein (supplementary Figures S9A–S9C), suggesting that HSCas9 promoted the CRISPR/Cas9 system to destroy HBV genome due to its higher stability.

Furthermore, we compared the efficacy of HSCas9/gHBV and entecavir (ETV) in inhibiting HBV replication. As shown in supplementary Figures S11A–S11E, the ability of HSCas9/gHBV to inhibit HBV replication was significantly higher than that of ETV, suggesting that HSCas9/gHBV has an advantage in clearing HBV infection.

To explore the off-target effects of CRISPR/Cas9 and CRISPR/HSCas9 systems in vivo, we performed whole-genome sequencing for genomic DNA extracted from liver tissues of 3 mice in each group. The results revealed that there were no significant differences in the numbers of SNP and indel variants among the three groups (Figures 6(E–H)), and no SNP and indel variants were found within 20 bp upstream and downstream of all potential off-target sites (supplementary Table S7), suggesting that HSCas9 did not increase off-target effects. To exclude the effects of cytotoxicity, haematoxylin–eosin (HE) staining of liver tissue was performed, and the levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in serum were detected. The results showed that both CRISPR/Cas9 and CRISPR/HSCas9 systems did not cause significant liver injury and inflammation (Figures 6(I,J), Supplementary Figure S12B).