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.

CRISPR-Cas9 mediated endogenous utrophin upregulation improves Duchenne Muscular Dystrophy

“…In addition, on-target DSB may lead to large deletions, complex rearrangements, chromothripsis or aneuploidy and chromosomal truncations (83)(84)(85). Hence more controlled novel and safer CRISPR-Cas9 based tools would be desirable in the future for translation (63). Since we demonstrated that a minimal modification of the mRNA Let-7c BS, e.g.…”

“…In summary, these data demonstrate that our CRISPR-Cas9 strategy targets utrophin 3'UTR with high efficiency and without major off-target effect. Additional studies to reduce offtargets (63) and indel quantification on edited muscles, normally exposed to lower gRNA/Cas9 concentration, will be performed before clinical translation.…”

CHANGE-seq-BE enables simultaneously sensitive and unbiasedin vitroprofiling of base editor genome-wide activity

“…Base editors can bind and modify off-target genomic loci with sequence homology to the target protospacer, similar to other genome editors 4–6 . As base editors rapidly advance towards clinical application, defining guide RNA (gRNA) dependent off-target activity of adenine base editors in a sensitive and unbiased way is important for ensuring the safety of these new therapeutic approaches 7 .…”

Gene editing ofCD3 epsilongene to redirect regulatory T cells for adoptive T cell transfer

“…The risk of off-target effects and other forms of genotoxicity is a concern when translating CRISPR-Cas to clinical protocols 31 . To confirm high specificity of our preferred CRISPR-AsCas12a gene editing strategy, we examined the off-target profile of the selected AsCas12a-crRNA #1 using CAST-Seq technology 32 which detects potential off-target sites by identification of translocations between the on-target and the putative off-target sites.…”