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

High-resolution crystal structures of three engineered Cas9 variants reveal altered PAM recognition.

• Wild-type Cas9 recognizes the 5'-NGG-3' PAM, while engineered variants VQR, EQR, and VRER recognize 5'-NGA-3', 5'-NGAG-3', and 5'-NGCG-3' PAMs, respectively.
• The structural comparison indicates that multiple mutations in the variants cause a displacement in the phosphodiester backbone of the PAM duplex.
• This displacement enables the SpCas9 variants to directly recognize altered PAM nucleotides.
• Findings provide insights into the mechanisms behind PAM specificity changes in Cas9 variants.
• Results may assist in the rational engineering of CRISPR-Cas9 for enhanced genome editing applications.

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