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

Singh, K; Evens, H; Nair, N; Rincón, MY; Sarcar, S; Samara-Kuko, E; Chuah, MK; (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. Green open access

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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.

Type: Article
Title: Efficient In Vivo Liver-Directed Gene Editing Using CRISPR/Cas9
Open access status: An open access version is available from UCL Discovery
DOI: 10.1016/j.ymthe.2018.02.023
Publisher version: https://doi.org/10.1016/j.ymthe.2018.02.023
Language: English
Additional information: © 2018 The Author(s). This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Keywords: CRISPR, Cas9, liver, gRNA, AAV, factor IX, hepatocytes, off-target, truncated gRNA
UCL classification: UCL
UCL > Provost and Vice Provost Offices
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences
URI: https://discovery.ucl.ac.uk/id/eprint/10062517
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