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

Abstract

CRISPR-Cas genome-editing nucleases hold substantial promise for developing human therapeutic applications but identifying unwanted off-target mutations is important for clinical translation. 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.

CRISPR/Cas Systems in Genome Editing: Methodologies and Tools for sgRNA Design, Off‐Target Evaluation, and Strategies to Mitigate Off‐Target Effects

“…VIVO is a very sensitive in vivo method which robustly detects off‐target effects caused by CRISPR/Cas nucleases in a genome‐wide scale . VIVO has been tested in vivo where a gRNA was deliberately designed with random sequence to check whether Cas9 can induce bona fide off‐targets in mouse livers.…”

“…VIVO demonstrated that correctly designed gRNAs can direct the efficient in vivo editing in mouse livers without any detectable off‐target mutations. VIVO is therefore suitable to define and quantify the off‐target mutations of Cas9 proteins in whole organisms …”

Epidermolysis bullosa: Advances in research and treatment

“…Whereas most CRISPR approaches harnessed the DNA double‐strand break inducing Streptococcus pyogenes Cas9 (SpCas9) nuclease variants, we have recently developed double nicking approaches based on single‐strand DNA breaks (nicks) at the targeting site with the intention to reduce off‐target site effects . Compared to the extremely high numbers of off‐target site modifications inherent to replacement gene therapies resulting from multiple random integrations in the treated cells, off‐target mutations are rare or non‐existent upon designer nuclease treatment …”

Paradoxical Survival: Examining the Parrondo Effect across Biology

“…Compounding onto this highly convoluted problem is our imperfect understanding of genetic dynamics at the current moment, especially concerning pleiotropic effects. Even if genetic editing, by clustered regularly interspaced short palindromic repeats (CRISPR) or other technologies, were to be free of off‐target errors—a goal towards which rapid progress is being made—great caution should be exercised in considering the eco‐evolutionary implications of our modifications. The enormous potential of biotechnology demands equivalent care and responsibility as research efforts continue.…”