Clustered, regularly interspersed, short palindromic repeat RNA-guided nucleases have emerged as highly efficient genome editing tools. However, in vivo tissue-specific genome editing at the desired loci is still a challenge. Here, we report that truncated guide RNAs (gRNAs) and Cas9 under the control of a computationally designed hepatocyte-specific promoter lead to liver-specific and target site-specific indel formation in the mouse factor IX (FIX) gene. The truncated gRNAs targeting unique sites in exon 1 and exon 6 of the mouse FIX gene were designed using a computational CRISPR design tool and the target sites overlap with mutations known to cause hemophilia B in patients. The gRNA and Cas9-expressing constructs were delivered in vivo using AAV9 vectors. The efficiency of in vivo targeting was assessed by T7E1 assays, site-specific Sanger sequencing and deep sequencing of on-target and putative off-target sites. 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. Consequently, the indel frequency was robust in the liver (up to 50%) in the desired target locus of the FIX gene, 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. Deep sequencing of putative off-target sites revealed no off-target editing. Our findings have potentially broad implications for somatic gene editing in the liver using the CRISPR/Cas9 platform.
|Number of pages||1|
|Publication status||Published - May 2016|
|Event||19th Annual Meeting of the American-Society-of-Gene-and-Cell-Therapy (ASGCT) - Washington DC, United States|
Duration: 4 May 2016 → 7 May 2016