CRISPR nuclease off-target activity and mitigation strategies.

Abstract

The discovery of CRISPR has allowed site-specific genomic modification to become a reality and this technology is now being applied in a number of human clinical trials. While this technology has demonstrated impressive efficacy in the clinic to date, there remains the potential for unintended on- and off-target effects of CRISPR nuclease activity. A variety of in silico-based prediction tools and empirically derived experimental methods have been developed to identify the most common unintended effect-small insertions and deletions at genomic sites with homology to the guide RNA. However, large-scale aberrations have recently been reported such as translocations, inversions, deletions, and even chromothripsis. These are more difficult to detect using current workflows indicating a major unmet need in the field. In this review we summarize potential sequencing-based solutions that may be able to detect these large-scale effects even at low frequencies of occurrence. In addition, many of the current clinical trials using CRISPR involve ex vivo isolation of a patient's own stem cells, modification, and re-transplantation. However, there is growing interest in direct, in vivo delivery of genome editing tools. While this strategy has the potential to address disease in cell types that are not amenable to ex vivo manipulation, in vivo editing has only one desired outcome-on-target editing in the cell type of interest. CRISPR activity in unintended cell types (both on- and off-target) is therefore a major safety as well as ethical concern in tissues that could enable germline transmission. In this review, we have summarized the strengths and weaknesses of current editing and delivery tools and potential improvements to off-target and off-tissue CRISPR activity detection. We have also outlined potential mitigation strategies that will ensure that the safety of CRISPR keeps pace with efficacy, a necessary requirement if this technology is to realize its full translational potential.