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

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 mous...

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Bibliographic Details
Published in:Molecular therapy 2018-05, Vol.26 (5), p.1241-1254
Main Authors: Singh, Kshitiz, Evens, Hanneke, Nair, Nisha, Rincón, Melvin Y., Sarcar, Shilpita, Samara-Kuko, Ermira, Chuah, Marinee K., VandenDriessche, Thierry
Format: Article
Language:English
Subjects:
AAV
Animals
Base Sequence
Binding Sites
Cas9
Coagulation factors
Computational Biology - methods
CRISPR
CRISPR-Cas Systems
Cytomegalovirus
Deoxyribonucleic acid
Dependovirus - genetics
DNA
Experiments
factor IX
Factor IX - genetics
Factor IX deficiency
Gene Editing
Gene expression
Gene Targeting
Genetic Vectors - genetics
Genome editing
Genomes
Genomics
Genotype & phenotype
gRNA
Hemophilia
Hemophilia B - diagnosis
Hemophilia B - genetics
Hemophilia B - therapy
hepatocytes
Humans
Liver
Liver - metabolism
Mice
Mutation
Neonates
off-target
Organ Specificity
Original
Phenotype
Phenotypes
Protein Binding
RNA, Guide, CRISPR-Cas Systems
Stem cells
truncated gRNA
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Summary: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.
ISSN:1525-0016
1525-0024
DOI:10.1016/j.ymthe.2018.02.023