An optimized SpCas9 high-fidelity variant for direct protein delivery

Area of Science:

• Molecular Biology
• Gene Editing Technologies

Background:

• Electroporation of Cas9 ribonucleoprotein (RNP) complexes prevents off-target cleavage and immune responses.
• Engineered high-fidelity Streptococcus pyogenes Cas9 (SpCas9) variants often have reduced activity and are incompatible with RNP delivery.

Purpose of the Study:

• To develop a high-fidelity SpCas9 variant suitable for RNP delivery.
• To compare the editing efficacy and precision of the novel recombinant high-fidelity Cas9 (rCas9HF) with the existing HiFi Cas9 (R691A mutant).

Main Methods:

• Developed rCas9HF (K526D substitution) based on previous evoCas9 studies.
• Compared rCas9HF and HiFi Cas9 (R691A) in RNP electroporation.
• Conducted gene substitution experiments using DNA donor templates to assess non-homologous end joining (NHEJ) versus homology-directed repair (HDR) ratios.

Main Results:

• rCas9HF is a high-fidelity SpCas9 variant compatible with RNP delivery.
• Comparative analysis revealed heterogeneous editing efficacy and precision between rCas9HF and HiFi Cas9 across the genome.
• The variants demonstrated different targeting capabilities and NHEJ/HDR ratios in gene editing experiments.

Conclusions:

• The development of rCas9HF provides a novel tool for precise genome editing.
• rCas9HF exhibits a distinct editing profile compared to HiFi Cas9 in RNP electroporation.
• This new variant expands genome editing solutions for highly precise and efficient applications.