Flexibility in PAM recognition expands DNA targeting in xCas9

Area of Science:

• Molecular Biology
• Genetics
• Biochemistry

Background:

• CRISPR-Cas9 is a powerful genome editing tool.
• Off-target effects and limited PAM recognition hinder its application.
• xCas9 is an engineered variant designed for enhanced specificity and broader targeting.

Purpose of the Study:

• To elucidate the molecular mechanism behind xCas9's expanded Protospacer Adjacent Motif (PAM) recognition.
• To understand how xCas9 achieves improved specificity and reduced off-target effects compared to wild-type Cas9.
• To provide insights for engineering future CRISPR-Cas systems with expanded DNA targeting capabilities.

Main Methods:

• Structural and biochemical analyses of xCas9 and wild-type Cas9.
• Investigating the role of specific amino acid residues, particularly R1335, in PAM recognition.
• Comparing DNA binding affinities and specificities of xCas9 and Cas9 to various PAM sequences.

Main Results:

• Wild-type Cas9's stringent guanine selection is mediated by a rigid arginine dyad.
• xCas9 introduces flexibility at R1335, enabling recognition of alternative PAM sequences.
• This flexibility confers an entropic preference, enhancing recognition of both canonical (TGG) and alternative PAMs.
• xCas9 shows dynamic binding preferences, favoring alternative PAMs early in evolution and canonical PAMs in the final stage.

Conclusions:

• The flexibility of the PAM-interacting cleft is crucial for expanding DNA targeting in CRISPR-Cas systems.
• xCas9's mechanism provides a blueprint for engineering CRISPR variants with broader PAM compatibility.
• Understanding xCas9's DNA recognition enhances the potential of CRISPR technology for diverse genomic applications.