Liver-directed gene editing with crispr-cas9: Comprehensive on-target and off-target analyses

Gene editing allows for the specific targeted modification of genes of living organisms or cells to improve our understanding of gene function and to use it to treat or cure diseases. Typically, gene editing requires the introduction of a double-strand DNA break (DSB) at a desired specific DNA target sequence while preventing undesired DSBs elsewhere in the genome. RNA-guided endonucleases based on the so-called ‘clustered regularly interspaced short palindromic repeats’ (CRISPR)/CRISPRassociated (Cas) mediated system can be used to achieve this type of DNA sequencespecific targeting. This DNA target sequence specificity exploits the principle of basepairing complementarity between the guide RNA (gRNA) and the target DNA sequence that subsequently guides the Cas9 endonuclease to its desired target DNA site. Ideally, CRISPR/Cas-mediated gene editing should only target the gene of interest without inducing any other genetic alterations at non-target sites in the genome, defined as “off-target” sites.
At the onset of this PhD thesis, there were relatively few studies examining the consequences of CRISPR/Cas-mediated in vivo gene editing. Given the important role of the liver in health and disease, we therefore specifically wanted to investigate the use of CRISPR/Cas to achieve gene-specific gene editing in hepatocytes in vivo. Ideally, liver-directed gene editing should only target the gene of interest without any off-target effects. However, this has not been thoroughly investigated since comprehensive studies are lacking regarding the on-target and off-target consequences of CRISPR/Cas-mediated in vivo gene targeting. We therefore wished to address the following research aims and hypotheses: (1) To assess whether in vivo delivery of the CRISPR-Cas system promotes efficient gene targeting at a target locus; (2) To assess the relative efficacy and specificity of different gRNAs designs; (3) To comprehensively assess off-target effects.
In this study, we therefore characterized the consequences of delivering CRISPR/Cas components with adeno-associated viral vectors (AAV) based on serotype 9 (AAV9). The guide RNAs were specifically designed to target the coagulation mouse factor IX (F9) gene as a clinically relevant target gene for hemophilia gene editing. Cas9 expression was controlled by a hepatocyte-specific promoter that lead to liver-specific and sequence-specific targeting of the mouse F9 gene. Though AAV9 transduction was apparent in multiple tissues and organs, Cas9 expression was restricted mainly to the liver, with only minimal or no expression in other non-hepatic tissues. The frequency of insertions/deletions (indels) in the liver at the F9 target locus was relatively robust (up to 50%), with no evidence of targeting in other organs. This resulted in a substantial loss of FIX activity and the emergence of a bleeding phenotype, consistent with hemophilia B. The gene targeting efficiency was similar with either full-length or truncated gRNA. Whole-genome sequencing (WGS) was subsequently conducted to examine off-target indels that were very rare or below the WGS detection limit. Shearing Extension Primer Tag Selection Ligation-Mediated PCR (S-EPTS/LM-PCR) revealed that the AAV vectors integrated preferentially into the CRISPR/Cas-induced DSB in the F9 gene. In contrast, no AAV integrations were detected in proximity of any of ∼5000 putative computationally predicted off-target sites (median distance of 70 kb). This is consistent with a low risk of off-target effects after in vivo liver-directed CRISPR/Cas-mediated gene editing that is likely due to the specific gRNA design and/or the transient expression of the CRISPR-Cas9 components in dividing neonatal hepatocytes. In conclusion, this PhD study underscores the potential of CRISPR/Cas technology for liver-directed gene-specific gene editing with minimal risk of potential off-target effects.