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 mutations limit CRISPR-Cas9's clinical applications.
• xCas9 is an engineered variant designed for enhanced specificity.

Purpose of the Study:

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

Main Methods:

• Comparative analysis of xCas9 and wild-type Cas9 PAM recognition.
• Structural and biochemical studies to investigate protein-DNA interactions.
• Evolutionary analysis of PAM sequence selection during xCas9 development.

Main Results:

• Wild-type Cas9's stringent guanine selection is due to a rigid arginine dyad.
• xCas9's R1335 flexibility allows recognition of alternative PAM sequences.
• This flexibility confers an entropic preference, improving canonical TGG PAM recognition.
• xCas9 initially favors alternative PAMs then shifts to canonical PAMs during evolution.

Conclusions:

• xCas9's expanded PAM recognition is mediated by increased flexibility in its PAM-interacting cleft.
• Fine-tuning cleft flexibility is a key strategy for broadening CRISPR-Cas DNA targeting.
• Understanding xCas9's mechanism offers insights applicable to other CRISPR-Cas systems.