Structural Basis for the Altered PAM Specificities of Engineered CRISPR-Cas9

Area of Science:

• Biochemistry
• Molecular Biology
• Structural Biology

Background:

• The CRISPR-Cas9 system is a powerful tool for genome editing.
• Wild-type Streptococcus pyogenes Cas9 (SpCas9) recognizes the 5'-NGG-3' protospacer adjacent motif (PAM).
• Engineered SpCas9 variants (VQR, EQR, VRER) exhibit altered PAM specificities, recognizing 5'-NGA-3', 5'-NGAG-3', and 5'-NGCG-3' respectively.

Purpose of the Study:

• To elucidate the structural basis for the altered PAM specificities of engineered SpCas9 variants.
• To provide a framework for the rational engineering of CRISPR-Cas9 systems.

Main Methods:

• High-resolution crystal structure determination.
• Complex formation of SpCas9 variants with single-guide RNA and altered PAM-containing DNA targets.

Main Results:

• The crystal structures revealed how SpCas9 variants recognize altered PAM sequences.
• Mutations in the variants synergistically displace the phosphodiester backbone of the PAM duplex.
• This displacement allows direct recognition of the altered PAM nucleotides.

Conclusions:

• The study explains the molecular mechanisms behind the altered PAM specificities of engineered SpCas9 variants.
• Findings facilitate the rational design of novel Cas9 variants for expanded genome editing applications.