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

Area of Science:

• Molecular Biology
• Genome Engineering
• Biotechnology

Background:

• CRISPR-Cas9 technology revolutionized genome engineering but is limited by protospacer-adjacent motif (PAM) recognition sequences.
• The Staphylococcus aureus Cas9 (SaCas9) enzyme is attractive for in vivo use due to its small size, but its NNGRRT PAM restricts genomic targeting.
• Existing SaCas9 variants, like KKH, were developed through experimental evolution to broaden PAM compatibility.

Purpose of the Study:

• To computationally design a novel SaCas9 variant (KRH) with an expanded PAM recognition profile.
• To evaluate the genome and base editing efficiency of KRH, particularly at non-canonical PAM sites.
• To elucidate the mechanistic basis for KRH's altered PAM specificity using computational modeling.

Main Methods:

• A fully computational point-mutation design workflow, UniDesign, was employed to engineer the SaCas9 variant KRH.
• KRH was characterized for its PAM recognition and editing efficiency without subsequent experimental optimization.
• Computational modeling, including structural and energetic analyses, was used to explain the observed PAM relaxation.

Main Results:

• The computationally designed KRH variant efficiently recognizes an expanded NNNRRT PAM.
• KRH demonstrated significantly enhanced editing efficiency at non-canonical PAM sites, with improvements up to 116-fold compared to wild-type SaCas9.
• KRH achieved editing efficiencies comparable to or exceeding those of the evolution-derived KKH variant.

Conclusions:

• KRH represents a rationally engineered SaCas9 variant with relaxed PAM specificity, expanding targeting capabilities for genome editing.
• Computational design, as exemplified by UniDesign, can effectively remodel Cas9 PAM interfaces and surpass evolution-derived variants.
• This work establishes KRH as a valuable tool and highlights computational design as a scalable strategy for developing next-generation CRISPR nucleases.