Rapid characterization of CRISPR-Cas9 protospacer adjacent motif sequence elements

Area of Science:

• Molecular Biology
• Genome Engineering
• Biotechnology

Background:

• Expanding the Cas9 toolbox is crucial for advancing genome targeting technologies.
• Understanding guide RNA and protospacer adjacent motif (PAM) interactions is key to Cas9 specificity.

Purpose of the Study:

• To develop a high-throughput in vitro method for characterizing Cas9-guide RNA and PAM specificities.
• To identify novel Cas9 orthologs with unique targeting capabilities.

Main Methods:

• In vitro cleavage assay using plasmid libraries with randomized PAM sequences.
• Varying Cas9-guide RNA complex concentrations to determine PAM preferences.
• Functional validation of identified Cas9s in vitro and in plant systems.

Main Results:

• The method accurately reproduced known PAM preferences for Streptococcus pyogenes, Sth3, and Sth1 Cas9s.
• Identified novel PAM and guide RNA specificities for a Cas9 from Brevibacillus laterosporus.
• Demonstrated the functional activity of the novel Brevibacillus laterosporus Cas9 in vitro and in plants.

Conclusions:

• The developed in vitro assay is effective for rapid screening of Cas9 requirements.
• This approach facilitates the discovery and characterization of new Cas9 variants for genome editing.
• The novel Brevibacillus laterosporus Cas9 expands the options for precise genome engineering.