CRISPR/Cas editing with no detectable genome-wide off-targets — method of validation

These GEITP pages have previously discussed CRISPR/Cas9, the new gene-editing methodology that has revolutionized molecular biology and molecular genetics studies –– especially in laboratory animal and plant model systems. Clinically, this type of gene-editing will be commonplace, as well. Very briefly summarized, CRISP/Cas9 allows the specific removal (cutting out one or more DNA nucleotides) and/or addition (insertion of one or more nucleotides) and/or replacement (substituting one or more different nucleotides) into the genome of an animal or plant. The possible downside of this technique has been that other (undesirable) DNA locations (so-called ‘off-targets’) might also be interfered with, and this error could lead to perturbations in some physiological function or even a disease, including cancer.

Authors [see attached article] describe ‘Verification of In Vivo Off-targets’ (VIVO), a highly sensitive strategy that can robustly identify any genome-wide off-target effects of CRISPR–Cas activity in the intact cell, tissue, or organism. VIVO consists of two steps. First, a superset of potential off-target cleavage sites for the nucleases is identified, using circularization for reporting cleavage effects in cell culture by sequencing (CIRCLE-seq); this sensitive method avoids potential confounding effects associated with cell-based assays and can successfully identify supersets of sites that include bona fide off-targets in cultured mouse or human cells. Second, sites identified by CIRCLE-seq are then examined for insertion-deletion (indel) mutations in target tissues that have been treated with Cas9 (which stands for ‘CRISPR-associated protein-9’, which is an RNA-guided DNA endonuclease enzyme).

Authors [see attached article] used VIVO, comparing it with a “guide RNA” (deliberately designed to be promiscuous) to show that CRISPR–Cas nucleases can induce substantial off-target mutations in mouse liver in the intact animal. Using VIVO, authors then show that appropriately designed guide RNAs can direct efficient editing in liver of the intact mouse with no detectable off-target mutations. Thus, VIVO provides a general strategy for defining and quantifying the off-target effects of gene-editing nucleases in whole organisms. This technique thereby provides a blueprint to foster the development of therapeutic strategies by which gene-editing in the intact cell or tissue can be used –– clinically, as well as in mouse.


Nature 20 Sept 2o18; 561: 416–419

This entry was posted in Center for Environmental Genetics. Bookmark the permalink.