Fully computational design of PAM-relaxed Staphylococcus aureus Cas9 with expanded targeting capability using...

Area of Science:

• Genetics and Genomics
• Protein Engineering
• Bioinformatics

Background:

• CRISPR-Cas9 gene editing is powerful but limited by protospacer-adjacent motif (PAM) recognition.
• Staphylococcus aureus Cas9 (SaCas9) is suitable for in vivo delivery but has a restrictive NNGRRT PAM.

Purpose of the Study:

• To develop a SaCas9 variant with relaxed PAM specificity using computational design.
• To assess the genome and base-editing efficiency of the designed variant.

Main Methods:

• Utilized UniDesign, a computational protein design workflow, to create SaCas9 variants.
• Introduced point mutations (E782K/N968R/R1015H) to generate the KRH variant.
• Evaluated editing efficiencies in multiple human cell types.

Main Results:

• The KRH variant efficiently recognizes an expanded NNNRRT PAM.
• KRH demonstrated genome- and base-editing efficiencies comparable to the KKH variant.
• Structural analysis revealed KRH fine-tunes interactions to relax PAM specificity.

Conclusions:

• Computational design can effectively engineer Cas9 variants with altered PAM specificity.
• The KRH variant offers a valuable tool for expanding genome editing applications.
• This approach provides a scalable strategy for developing novel Cas9 nucleases.