A system to effectively identify genome-wide off-target effects from CRISPR-Cas9 genome editing has been tested in mice in a paper published online this week in Nature. These findings could facilitate research into the clinical translation of genome editing.
CRISPR-Cas9 genome editing is seen as a promising potential innovation in human medicine, but unwanted additional (off-target) mutations must be identified and prevented before the technique can progress towards clinical use. Off-target mutations occur when the CRISPR-Cas9 complex attaches to multiple sites in the genome and cuts the wrong section of DNA. Systems to detect these mutations must be tested in whole organisms as well as cells.
J. Keith Joung, Marcello Maresca, and colleagues describe 'verification of in vivo off-targets' (VIVO), a highly sensitive strategy that can identify off-target mutations from CRISPR-Cas9 in a living organism. VIVO involves first identifying potential off-target sites, and then confirming whether any such sites have been altered after genome editing has occurred. The authors tested the system’s precision in mouse livers by designing different guide RNAs (gRNAs) targeted to the mouse Pcsk9 gene, including promiscuous (those able to target many sites) and more specific gRNAs. They demonstrated that VIVO could not only detect the dozens of off-target mutations induced by the promiscuous gRNA (including mutations that occurred with frequencies as low as 0.13%) but that appropriately designed gRNAs did not show any detectable off-target mutations.
The authors suggest that VIVO sets an important standard for defining the off-target effects of genome editing in practice, and demonstrates the importance of designing gRNAs to be as specific as possible.