Impact of Genetic Variation on CRISPR-Cas Targeting

Abstract

The CRISPR-CRISPR-associated (Cas) nuclease system offers the ability to perform unprecedented functional genetic experiments and the promise of therapy for a variety of genetic disorders. The understanding of factors contributing to CRISPR targeting efficacy and specificity continues to evolve. As CRISPR systems rely on Watson-Crick base pairing to ultimately mediate genomic cleavage, it logically follows that genetic variation would affect CRISPR targeting by increasing or decreasing sequence homology at on-target and off-target sites or by altering protospacer adjacent motifs. Numerous efforts have been made to document the extent of human genetic variation, which can serve as resources to understand and mitigate the effect of genetic variation on CRISPR targeting. Here, we review efforts to elucidate the effect of human genetic variation on CRISPR targeting at on-target and off-target sites with considerations for laboratory experiments and clinical translation of CRISPR-based therapies.

FIG. 1.

Alteration of on-target and off-target sites by variants. (a) On-target variation resulting in gRNA activity attenuation or complete loss of gRNA activity as assessed by CFD scores. Genomic sequences with mismatches to the gRNA are highlighted in red. Bolded bases indicate bases that are altered in the corresponding reference or nonreference genome. Vertical lines indicate bonds between the gRNA and the cognate homologous sequence at the on-target site. (b) Off-target variation resulting in alteration of the potency of off-target sites as well as PAM creation or PAM destruction as assessed by CFD scores. Genomic sequences with mismatches to the gRNA are highlighted in red. Bolded bases indicate bases that are altered in the corresponding reference or nonreference genome. Vertical lines indicate bonds between the gRNA and the cognate homologous sequence at off-target sites. CFD, cutting frequency determination; gRNA, guide RNA; PAM, protospacer adjacent motif.

FIG. 2.

Hypothetical patient-specific sequences for different individuals seeking CRISPR-based therapeutic genome editing that may be prone to treatment failure and/or adverse outcomes due to genetic variation as assessed by CFD. Genomic sequences with mismatches to the gRNA are highlighted in red. Bolded bases indicate bases that are altered in the genome of another patient. Vertical lines indicate bonds between the gRNA and the cognate homologous sequence at on-target or off-target sites.