Decoding CRISPR-Cas PAM recognition with UniDesign.

Area of Science:

• Biochemistry
• Molecular Biology
• Bioinformatics

Background:

• CRISPR-Cas gene editing relies on specific DNA recognition.
• Protospacer adjacent motifs (PAMs) are crucial for target DNA binding by CRISPR-Cas proteins.
• Understanding PAM-interacting amino acid (PIAA) interactions is key to engineering CRISPR-Cas systems.

Purpose of the Study:

• To develop a universal computational protein design framework (UniDesign) for modeling protein-nucleic acid interactions.
• To decode the PAM-PIAA interactions for various Cas proteins.
• To demonstrate UniDesign's utility in engineering CRISPR-Cas systems.

Main Methods:

• Application of the UniDesign framework to eight Cas9 and two Cas12a proteins.
• Decoding PAM recognition by analyzing PIAA sequences.
• Computational prediction and redesign of PAM sequences and PIAA residues.

Main Results:

• UniDesign accurately predicted native PAMs when provided with native PIAAs for all tested Cas proteins.
• Computationally redesigned PIAAs largely recapitulated native PIAAs when provided with natural PAMs (74% identity, 86% similarity).
• UniDesign effectively captures the mutual preference between natural PAMs and native PIAAs.

Conclusions:

• UniDesign is a robust tool for understanding and engineering protein-nucleic acid interactions.
• The framework shows promise for modifying CRISPR-Cas systems to alter PAM specificity.
• UniDesign can be applied to engineer other nucleic acid-interacting proteins.