VIVO Method Validates CRISPR In Vivo Editing with No Detectable Off-Target Mutations
Researchers developed VIVO (verification of in vivo off-targets), a highly sensitive genome-wide strategy to detect off-target CRISPR-Cas nuclease activity in living organisms. Using mouse liver editing, they demonstrated that well-designed guide RNAs can achieve efficient on-target editing with no detectable off-targets, while deliberately promiscuous guides produce substantial unwanted mutations. VIVO provides a critical quality control framework for clinical gene-editing programs requiring comprehensive safety assessment.
The original study
In vivo CRISPR editing with no detectable genome-wide off-target mutations.
- Authors
- Akcakaya P, Bobbin ML, Guo JA, Malagon-Lopez J, Clement K, Garcia SP, et al.
- Journal
- Nature
- Type
- Journal Article, Research Support, N.I.H., Extramural, Research Support, Non-U.S. Gov't, Research Support, U.S. Gov't, Non-P.H.S., Validation Study
- PMID
- 30209390
Original abstract
CRISPR-Cas genome-editing nucleases hold substantial promise for developing human therapeutic applications1-6 but identifying unwanted off-target mutations is important for clinical translation7. A well-validated method that can reliably identify off-targets in vivo has not been described to date, which means it is currently unclear whether and how frequently these mutations occur. Here we describe 'verification of in vivo off-targets' (VIVO), a highly sensitive strategy that can robustly identify the genome-wide off-target effects of CRISPR-Cas nucleases in vivo. We use VIVO and a guide RNA deliberately designed to be promiscuous to show that CRISPR-Cas nucleases can induce substantial off-target mutations in mouse livers in vivo. More importantly, we also use VIVO to show that appropriately designed guide RNAs can direct efficient in vivo editing in mouse livers with no detectable off-target mutations. VIVO provides a general strategy for defining and quantifying the off-target effects of gene-editing nucleases in whole organisms, thereby providing a blueprint to foster the development of therapeutic strategies that use in vivo gene editing.