Efficient In Vivo Liver-Directed Gene Editing Using CRISPR/Cas9

Kshitiz Singh, Hanneke Evens, Nisha Nair, Melvin Y Rincón, Shilpita Sarcar, Ermira Samara-Kuko, Marinee K Chuah, Thierry VandenDriessche

Research output: Contribution to journalArticlepeer-review

47 Citations (Scopus)


In 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. Efficient organ-specific gene targeting by CRISPR/Cas remains challenging, especially with large Cas9 variants. Singh et al. now demonstrate efficient hepatocyte-specific gene inactivation in adult or newborn mice using truncated guide RNAs in combination with a robust de novo-designed hepatocyte-specific promoter to drive SpCas9, with no demonstrable off-target effects.

Original languageEnglish
Pages (from-to)1241-1254
Number of pages14
JournalMolecular Therapy
Issue number5
Early online date2018
Publication statusPublished - 2 May 2018


  • AAV
  • Cas9
  • factor IX
  • gRNA
  • hepatocytes
  • liver
  • off-target
  • truncated gRNA


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