Singh, K; Evens, H; Nair, N; Rincón, MY; Sarcar, S; Samara-Kuko, E; Chuah, MK; Singh, K; Evens, H; Nair, N; Rincón, MY; Sarcar, S; Samara-Kuko, E; Chuah, MK; VandenDriessche, T; - view fewer (2018) Efficient In Vivo Liver-Directed Gene Editing Using CRISPR/Cas9. Molecular Therapy , 26 (5) pp. 1241-1254. 10.1016/j.ymthe.2018.02.023.

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Abstract

n vivo tissue-specific genome editing at the desired loci is still a challenge. Here, we report that AAV9-delivery of truncated guide RNAs (gRNAs) and Cas9 under the control of a computationally designed hepatocyte-specific promoter lead to liver-specific and sequence-specific targeting in the mouse factor IX (F9) gene. 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 insertions and deletion (indel) frequency was robust in the liver (up to 50%) in the desired target loci of the F9 gene, with no evidence of targeting in other organs or other putative off-target sites. This resulted in a substantial loss of FIX activity and the emergence of a bleeding phenotype, consistent with hemophilia B. The in vivo efficacy of the truncated gRNA was as high as that of full-length gRNA. Cas9 expression was transient in neonates, representing an attractive “hit-and-run” paradigm. Our findings have potentially broad implications for somatic gene targeting in the liver using the CRISPR/Cas9 platform.

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