In vivo CRISPR editing with no detectable genome-wide off-target mutations

CRISPR–Cas genome-editing nucleaseshold 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 vivohas not been described to date, which means it is currently unclear whether and how frequently these mutations occur. Here we describe ‘verification of in vivooff-targets’ ( VIVO), a highly sensitive strategy that can robustly identify the genome-wide off-target effects of CRISPR–Cas nucleasesin vivo. We use VIVOand a guide RNAdeliberately designed to be promiscuousto show that CRISPR–Cas nucleasescan induce substantial off-target mutations in mouse livers in vivo. More importantly, we also use VIVOto show that appropriately designed guide RNAscan direct efficient in vivoediting in mouse livers with no detectable off-target mutations. VIVOprovides a general strategy for defining and quantifying the off-target effects of gene-editing nucleasesin whole organisms, thereby providing a blueprint to foster the development of therapeutic strategies that use in vivogene editing.