biostudies-literatureHuang XUniversity of MichiganCystic Fibrosis FoundationNHLBI NIH HHSNational Institutes of HealthNIGMS NIH HHSbbad133https://www.ebi.ac.uk/biostudies/studies/S-EPMC10199764biostudies-literatureUnknown24(3)The critical first step in Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated (CRISPR-Cas) protein-mediated gene editing is recognizing a preferred protospacer adjacent motif (PAM) on target DNAs by the protein's PAM-interacting amino acids (PIAAs). Thus, accurate computational modeling of PAM recognition is useful in assisting CRISPR-Cas engineering to relax or tighten PAM requirements for subsequent applications. Here, we describe a universal computational protein design framework (UniDesign) for designing protein-nucleic acid interactions. As a proof of concept, we applied UniDesign to decode the PAM-PIAA interactions for eight Cas9 and two Cas12a proteins. We show that, given native PIAAs, the UniDesign-predicted PAMs are largely identical to the natural PAMsBriefings in bioinformaticsDecoding CRISPR-Cas PAM recognition with UniDesign.PMC10199764R41 GM122181R01 HL147527HL159871R01 HL159900R42 GM122181XU19XX0GM122181R01 HL159871HL147527HL159900R41 GM149016GM149016Xia XZhang JChen YEHuang XYang DXu JZhou JfalseDecoding CRISPR-Cas PAM recognition with UniDesign.The critical first step in Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated (CRISPR-Cas) protein-mediated gene editing is recognizing a preferred protospacer adjacent motif (PAM) on target DNAs by the protein's PAM-interacting amino acids (PIAAs). Thus, accurate computational modeling of PAM recognition is useful in assisting CRISPR-Cas engineering to relax or tighten PAM requirements for subsequent applications. Here, we describe a universal computational protein design framework (UniDesign) for designing protein-nucleic acid interactions. As a proof of concept, we applied UniDesign to decode the PAM-PIAA interactions for eight Cas9 and two Cas12a proteins. We show that, given native PIAAs, the UniDesign-predicted PAMs are largely identical to the natural PAMs2023-01-01T00:00:00Z2023 May2026-06-01T21:19:41.461Z2025-06-27T03:05:16.757ZS-EPMC101997643707868810.1093/bib/bbad133