Methods for Optimizing CRISPR-Cas9 Genome Editing Specificity

Abstract

Summary Advances in the development of delivery, repair, and specificity strategies for the CRISPR-Cas9 genome engineering toolbox are helping researchers understand gene function with unprecedented precision and sensitivity. CRISPR-Cas9 also holds enormous therapeutic potential for the treatment of genetic disorders by directly correcting disease-causing mutations. Although the Cas9 protein has been shown to bind and cleave DNA at off-target sites, the field of Cas9 specificity is rapidly progressing with marked improvements in guide RNA selection, protein and guide engineering, novel enzymes, and off-target detection methods. We review important challenges and breakthroughs in the field as a comprehensive practical guide to interested users of genome editing technologies, highlighting key tools and strategies for optimizing specificity. The genome editing community should now strive to standardize such methods for measuring and reporting off-target activity, while keeping in mind that the goal for specificity should be continued improvement and vigilance.

CRISPR–Cas9 Structures and Mechanisms

“…Abbreviations: bp, base pair; NUC, nuclease lobe; PAM, protospacer adjacent motif; REC, recognition lobe; sgRNA, single-guide RNA. subtype of Cas9-based CRISPR systems have been implemented for genome engineering in eukaryotes (22,39,98 subtype II-B Cas9 from Francisella novicida (FnCas9) consists of 1,629 amino acids and is significantly larger than other Cas9 orthologs from II-A and II-C subtypes (23,85). In addition to divergent lengths and sequences, Cas9 orthologs recognize distinct dual-RNA and PAM sequences for their functionality (23,59).…”

Designing Epigenome Editors: Considerations of Biochemical and Locus Specificities

“…These mutations weaken eCas9 binding to the non-target strand allowing the non-target and target DNA strands to re-hybridize more easily. The results of these studies demonstrate a significant reduction in genome wide off-target nuclease activity due to the increased dependence on the binding energy provided by the Watson-Crick base pairing between the gRNA and the target strand [119, 121]. In addition, eCas9 is reported to have comparable on-target cleavage activity with wild type Cas9.…”

“…In addition, eCas9 is reported to have comparable on-target cleavage activity with wild type Cas9. This feature could make this new system superior to other strategies to improve Cas9 specificity, because it increases specificity without sacrificing efficiency, although any impact on epigenome modifications should be confirmed [121]. Applicability of this elegant approach to dCas9 binding in epigenome editing studies needs to be investigated by future studies.…”

Improving the Function of Crispr-Cas9 for Genome Editing Therapy: Editing the Editor

“…Indeed, the researchers found multiple off-sites undetected with existing bioinformatics computational tools or ChIP-seq (Gabriel et al 2015). Other techniques for direct offtarget detection include integration-deficient lentiviral vector, BLESS, HTGTS, and Digenome-seq (reviewed in Tycko et al 2016).…”