Specifically targeting HBV DNA enabling deactivation or elimination using the CRISPR/Cas9 system is an attractive goal aimed to cure HBV. Selecting effective and specific target sequences in the viral DNA genome to design the HBV-specific sgRNA is the first and critical step in constructing the CRISPR/Cas9 system. In published studies, various target sites in the HBV genome, including the four ORFs (C, P, S and X), were selected as targets in designing HBV-specific sgRNA. To avoid off-target effects and minimize toxicity, it is necessary to use conserved sequences in the HBV genome and avoid similar parts of the human genome. Based on studies published by September 2015, HBV antigens, its DNA genome and cccDNA were all found to be significantly suppressed by certain HBV-specific gRNAs during in vitro or in vivo experiments (Table 1) (Lin et al., 2014; Seeger and Sohn, 2014; Dong et al., 2015; Karimova et al., 2015; Kennedy et al., 2015a; Liu et al., 2015; Ramanan et al., 2015; Wang et al., 2015; Zhen et al., 2015)
Table 1. Published studies on the application of CRISPR/Cas9 system to inhibit HBV replication.
In detail, it was reported that transfection of Cas9/ sgRNAs into cultured cells decreased HBV DNA release by 77% to 98% with different sgRNAs (Ramanan et al., 2015). The total amount of cccDNA was shown to decrease by 60.6% to 75.0% in Huh7 cells transfected with pCas9 (Dong et al., 2015). Kennedy's group reported that HBV RT-specific sgRNA repressed cccDNA formation by 10-fold, while surface Ag and core-specific sgRNAs sup pressed cccDNA levels by 4-fold (Kennedy et al., 2015a). Lin et al. used a duck hepatitis B virus (DHBV)-expressing plasmid and found that DHBV-specific gRNAs only slightly reduced the levels of the DHBV-expressing plasmid but efficiently suppressed the levels of rcDNA and cccDNA (Lin et al., 2014). In vivo, immunoblots revealed that only a minimal amount of the intracellular core protein was produced in pCas-injected mouse liver. Quantification of intrahepatic cccDNA also showed a reduction on CRISPR/Cas targeting (Dong et al., 2015).
Of note, various genome targeted gRNA/Cas9 systems showed various suppression efficiencies on HBV DNA and antigen expression. For example, surface Ag-specific sgRNA suppressed HBsAg production more effectively than other sgRNAs, with the inhibition rate ranging from 60% to almost 100% (HBsAg undetectable) with different cell culture systems and transfection methods (Lin et al., 2014; Kennedy et al., 2015a). Cells transduced with RT specific sgRNA showed a statistically significant reduction in HBeAg secretion. In contrast, there was no significant reduction in HBeAg for core and surface sgRNAs (Kennedy et al., 2015a). Dong et al. (2015) and Lin et al. (2014) reported that X region specific sgRNA exhibited powerful CRISPR/Cas9-mediated inhibition of HBV, perhaps because X encodes a protein that regulates viral gene transcription and is required for efficient viral replication and spread (Tang et al., 2006). The P region was another efficient site for specific targeting of sgRNA in suppressing HBV DNA replication and antigen transcription. Results from Kennedy et al. showed that RT-specific sgRNA, which targets the essential "YMDD" motif in the HBV P ORF for cleavage, essentially entirely blocked virus replication, shown by a reduction in total viral DNA released into the culture medium of 1000-fold, total intracellular HBV DNA levels decreased by 100-fold, and inhibition of the accumulation of cccDNA by up to 10-fold (Kennedy et al., 2015a).
One advantage of CRISPR/Cas9 compared with other gene editing methods is that it has the capacity for multiplex targeting by providing a method for multiple disruptions, insertions, and deletions with high efficiency and low cost. Therefore, the combination of different specific site sgRNAs targeting several sequences in the HBV genome for multiple genomic editing will be an ideal way to improve the suppression efficiency. Lin et al. reported that the combination of sgRNAs P1 and XCp was more effective in suppressing intracellular HBsAg production than either sgRNA alone (Lin et al., 2014). In vivo, the in-hibitory effect of CRISPR/Cas9 on serum HBsAg levels was highest (93%) for a combination of X and S specific sgRNAs (Zhen et al., 2015). However, combination of multiple sgRNAs also produced side-effects in the results of Wang et al., who found that the specific DNA fragment between the two cleavage sites of the gRNAs was removed by use of the dual gRNAs (Wang et al., 2015). Based on the fact that specific sgRNAs and combinations may result in differences in editing efficiency, strategies for specific HBV region sgRNA design and combination should be precisely verified in future studies.
In addition to cleavage, functional inactivation of HBV DNA (in particular cccDNA) caused by mutation also contributes to the inhibitory effects of CRISPR/Cas9 in suppressing HBV replication. Cas9 cleavage of targets in the residual viral DNA usually results in the introduction of small sequence insertions or deletions (indels), which can be assessed by T7 endonuclease (T7E1) assay and DNA sequencing. Dong et al. reported that an amplified PCR fragment of cccDNA was cleaved into 240 and 540 bp pieces in pCas9-1-transfected cells and into 380 and 400 bp pieces in pCas9-2-transfected Huh7 cells co-transfected with HBV precursor plasmid precccDNA, judged by T7E1 assay (Dong et al., 2015). Another study in Huh7 cells transfected with vector pAAV/HBV1.2 reported that the mutation rate in HBV expression templates edited by sgRNAs was 9.3%-13.6% with single sgRNAs, and up to 25.6% with a combination of sgRNAs (Lin et al., 2014). When sequencing DNA fragments amplified in the T7E1 assay, 58%–75% of the amplified HBV sequences contained mutations in S-and X-sgRNA/hCas9-treated mice (Zhen et al., 2015). The most frequent mutations (66%) were single-nucleotide deletions or insertions, followed by deletions that spanned > 100 nucleotides (19%), then deletions of < 30 nucleotides (12%) (Seeger and Sohn, 2014). These mutation patterns of HBV induced by CRISPR/Cas9 are observed as a result of DNA repair in the non-homologous end joining (NHEJ) pathway and typically occurred after genome editing(Cong et al., 2013; Seeger and Sohn, 2014; Dong et al., 2015; Zhen et al., 2015). In contrast, up to now no study showed that HDR was involved in repairing the HBV cccDNA cleaved by CRISPR/Cas9. However, with the introduction of homologous DNA sequence, HDR can be expected as a method in CRISPR/Cas9 system to specifically define HBV DNA at planed sites, such as sequence substitution, deletion and insertion (Figure 2).
Interestingly, although the mutation rates of the HBV genome are modest in vivo, the decrease in HBV DNA is quite remarkable in these studies. This inconsistency of mutation and clearance rates of viral templates might be attributed to fragments of HBV template cleaved by sgRNA/Cas9 not being completely rejoined by NHEJ; most of them may be cleared by other undefined mechanisms (Liu et al., 2015).
Current therapeutic treatments for chronic HBV infection include the use of INF-α and NAs, such as lamivudine (3TC), tenofovir disoproxil fumarate (TDF) and entecavir (ETV) (Koumbi, 2015). The fact that they cannot eliminate HBV replication completely has prompted the search for combination therapies using CRISPR/Cas9 with NAs or IFN-α, aimed at eliminating latent viral reservoirs. Experimental results show that IFN-α neither has a measurable effect on the antiviral activity of the CRISPR/ Cas9 system, nor affects the mutations of cccDNA (Seeger and Sohn, 2014). In contrast, treatment of HepG2.2.15 cells with HBV specific sgRNA transfection as well as TDF, ETV, or 3TC demonstrated a clear effect, at least additive, leading to more efficient elimination of residual HBV DNA replication. Significantly, the residual HBV DNA after the use of ETV was almost completely inhibited on application of the CRISPR/Cas9 system, which further confirms the potential of this approach to very efficiently edit cccDNA (Kennedy et al., 2015a). Thus, CRISPR/Cas9 might complement the presently available HBV antiviral treatment regimens, especially NAs, providing therapeutic approaches against targets that include HBV latent viral reservoirs of cccDNA.