Engineering Cas9: next generation of genomic editors.

The Cas9 endonuclease of the CRISPR/Cas type IIA system from Streptococcus pyogenes is the heart of genome editing technology that can be used to treat human genetic and viral diseases. Despite its large size and other drawbacks, S. pyogenes Cas9 remains the most widely used genome editor. A vast amount of research is aimed at improving Cas9 as a promising genetic therapy. Strategies include directed evolution of the Cas9 protein, rational design, and domain swapping. The first generation of Cas9 editors comes directly from the wild-type protein. The next generation is obtained by combining mutations from the first-generation variants, adding new mutations to them, or refining mutations. This review summarizes and discusses recent advances and ways in the creation of next-generation genomic editors derived from S. pyogenes Cas9. KEY POINTS: • The next-generation Cas9-based editors are more active than in the first one. • PAM-relaxed variants of Cas9 are improved by increased specificity and activity. • Less mutagenic and immunogenic variants of Cas9 are created.

Improving the on-target activity of high-fidelity Cas9 editors by combining rational design and random mutagenesis.

Genomic and post-genomic editors based on CRISPR/Cas systems are widely used in basic research and applied sciences, including human gene therapy. Most genome editing tools are based on the CRISPR/Cas9 type IIA system from Streptococcus pyogenes. Unfortunately, a number of drawbacks have hindered its application in therapeutic approaches, the most serious of which is the relatively high level of off-targets. To overcome this obstacle, various high-fidelity Cas9 variants have been created. However, they show reduced on-target activity compared to wild-type Cas9 possibly due to increased sensitivity to eukaryotic chromatin. Here, we combined a rational approach with random mutagenesis to create a set of new Cas9 variants showing high specificity and increased activity in Saccharomyces cerevisiae yeast. Moreover, a novel mutation in the PAM (protospacer adjacent motif)-interacting Cas9 domain was found, which increases the on-target activity of high-fidelity Cas9 variants while retaining their high specificity. The obtained data suggest that this mutation acts by weakening the eukaryotic chromatin barrier for Cas9 and rearranging the RuvC active center. Improved Cas9 variants should further advance genome and post-genome editing technologies. KEY POINTS: • D147Y and P411T mutations increase the activity of high-fidelity Cas9 variants. • The new L1206P mutation further increases the activity of high-fidelity Cas9 variants. • The L1206P mutation weakens the chromatin barrier for Cas9 editors.

Multiplex nucleotide editing by high-fidelity Cas9 variants with improved efficiency in rice.

BACKGROUND: Application of the CRISPR/Cas9 system or its derived base editors enables targeted genome modification, thereby providing a programmable tool to exploit gene functions and to improve crop traits. RESULTS: We report that PmCDA1 is much more efficient than rAPOBEC1 when fused to CRISPR/Cas9 nickase for the conversion of cytosine (C) to thymine (T) in rice. Three high-fidelity SpCas9 variants, eSpCas9(1.1), SpCas9-HF2 and HypaCas9, were engineered to serve with PmCDA1 (pBEs) as C-to-T base editors. These three high-fidelity editors had distinct multiplex-genome editing efficiencies. To substantially improve their base-editing efficiencies, a tandemly arrayed tRNA-modified single guide RNA (sgRNA) architecture was applied. The efficiency of eSpCas9(1.1)-pBE was enhanced up to 25.5-fold with an acceptable off-target effect. Moreover, two- to five-fold improvement was observed for knock-out mutation frequency by these high-fidelity Cas9s under the direction of the tRNA-modified sgRNA architecture. CONCLUSIONS: We have engineered a diverse toolkit for efficient and precise genome engineering in rice, thus making genome editing for plant research and crop improvement more flexible.