CRISPR/Cas9 searches for a protospacer adjacent motif by lateral diffusion

Area of Science:

• Molecular Biology
• Biochemistry
• Genetics

Background:

• The CRISPR/Cas9 system from Streptococcus pyogenes (SpCas9) is a powerful tool for genome editing.
• The precise molecular mechanisms underlying Cas9's DNA search and target recognition remain incompletely understood.
• Investigating transient Cas9-DNA interactions at high spatio-temporal resolution is essential to clarify these mechanisms.

Purpose of the Study:

• To elucidate the real-time dynamics of Cas9-DNA interactions during target searching.
• To investigate the role of protospacer adjacent motif (PAM) recognition in Cas9's search strategy.
• To develop a high-resolution method for observing transient molecular interactions.

Main Methods:

• Development and application of single-molecule Förster resonance energy transfer (smFRET) assays.
• Real-time monitoring of transient interactions between SpCas9 and DNA molecules.
• Analysis of Cas9's diffusion and binding dynamics in relation to PAM sequences.

Main Results:

• SpCas9 exhibits weak initial interactions with PAM sequences.
• Cas9 utilizes facilitated diffusion to explore neighboring DNA sequences.
• This dynamic exploration facilitates translocation to nearby PAM sites, leading to on-target binding.
• A model is proposed where lateral diffusion competes with 3D diffusion in PAM finding.

Conclusions:

• Cas9 employs a dynamic search mechanism involving facilitated diffusion and lateral exploration for PAM recognition.
• The findings provide insights into the spatio-temporal aspects of Cas9-DNA interactions.
• Neighboring sequences play a significant role in guiding Cas9 to its target, impacting genetic engineering strategies.