Evaluation and Reduction of CRISPR Off-Target Cleavage Events

Abstract

Introduction of CRISPR/Cas9 methods (clustered regularly interspaced short palindromic repeats, CRISPR-associated protein 9) have led to a huge surge in the use of precision genome editing for research applications. Translational medical efforts are likewise rapidly progressing, and Phase I clinical trials using these techniques have already started. As with any new technology that is applied to medical therapeutics, risks must be carefully defined and steps taken to mitigate side effects wherever possible. Effective methods are now available that permit identification of off-target cleavage events, a major class of potential side effects seen in mammalian genome editing. Off-target prediction algorithms are improving and have utility, but are insufficient to use alone. Empiric methods to define the off-target profile must also be used. Once defined, the frequency of off-target cleavage can be minimized using methods that limit the duration of exposure of the genome to the active genome editing complex, for example, using the ribonucleoprotein (RNP) approach. In addition, Cas9 mutants have been developed that markedly reduce the rate of off-target cleavage compared to the wild-type enzyme. Use of these new tools should become standard practice for medical applications.

Keywords: AAV; CRISPR/Cas9; gene editing; hematopoietic stem cells.