A highly specific SpCas9 variant is identified by in vivo screening in yeast

Abstract

Despite the utility of CRISPR-Cas9 nucleases for genome editing, the potential for off-target activity limits their application, especially for therapeutic purposes1,2. We developed a yeast-based assay to identify optimized Streptococcus pyogenes Cas9 (SpCas9) variants that enables simultaneous evaluation of on- and off-target activity. We screened a library of SpCas9 variants carrying random mutations in the REC3 domain and identified mutations that increased editing accuracy whilst maintaining editing efficiency. We combined four beneficial mutations to generate evoCas9, a variant that has fidelity exceeding both wild-type (79-fold improvement) and rationally designed Cas9 variants3,4 (4-fold average improvement), while maintaining near wild-type on-target editing efficiency (90% median residual activity). Evaluating evoCas9 on endogenous genomic loci, we demonstrated a substantial improved specificity and observed no off-target sites for 4 of the 8 sgRNAs tested. Finally, we showed that following long-term expression (40 days), evoCas9 strongly limited the unspecific cleavage of a difficult-to-discriminate off-target site and fully abrogated the cleavage of two additional off-targets.

CRISPR/Cas Systems in Genome Editing: Methodologies and Tools for sgRNA Design, Off‐Target Evaluation, and Strategies to Mitigate Off‐Target Effects

“…For example, the cleavage activity of Cas 9 could be blocked by two bacteriophage proteins AcrIIA2, AcrIIA4 (anti‐CRISPR proteins) after Cas9 cutting the target specific region . In recent years, serial Cas9 variants with high fidelity have been developed through optimizing and delineating the structure of Cas9 including eSpCas9(1.1), SpCas9‐HF1, Sniper‐Cas9, HypaCas9, xCas9(3.7), evoCas9, and SpCas9‐NG …”

A CRISPR–Cas9 system for multiple genome editing and pathway assembly in Candida tropicalis

“…To further validate the system, two additional genes, ADE2 (encoding phosphoribosylaminoimidazole carboxylase) and CAT , were targeted. Yeast carrying an ADE2 disruption display a distinct red colony color phenotype (Casini et al, ; Vyas et al, ), whereas CAT knockout strains are unable to grow when n ‐alkanes are used as the sole carbon source; instead, they accumulate a high amount of α,ω‐dodecanedioic acid (DCA12; L. Zhang et al, ). When the transient CRISPR–Cas9 system was used to target the CAT alleles with the cassette “ CAT1 ‐ gda324 ‐ URA3 ‐ CAT1 ” as the homologous repair template, almost 57% of randomly selected prototrophic colonies have the “ cat::gda324 ‐ URA3 / cat::gda324 ‐ URA3 ” genotype.…”

“…(encoding phosphoribosylaminoimidazole carboxylase) and CAT, were targeted. Yeast carrying an ADE2 disruption display a distinct red colony color phenotype (Casini et al, 2018;Vyas et al, 2015), whereas CAT knockout strains are unable to grow when n-alkanes are used as the sole carbon source; instead, they accumulate a high amount of α,ω-dodecanedioic acid (DCA12; L. Zhang et al, 2016).…”

Massively parallel CRISPRi assays reveal concealed thermodynamic determinants of dCas12a binding

“…Our multiplexed approach is particularly applicable to the advancement of dCas-based gene circuit elements, which can be been used to create complex circuits that behave orthogonally, operating independently without crosstalk [47][48][49][50][51]. Furthermore, our approach can expedite the rational design of enhanced CRISPR nucleases and facilitate the development of CRISPR-Cas variants with greater specificity, improved proofreading capabilities, or increased activities [52][53][54][55][56][57].…”