Highly active CRISPR-adaptation proteins revealed by a robust enrichment technology

Area of Science:

• Molecular Biology
• Microbial Genetics

Background:

• The CRISPR-Cas system provides prokaryotic immunity through spacer acquisition.
• Adaptation, a key step in CRISPR immunity, involves integrating foreign DNA spacers into the CRISPR array.
• Enhancing adaptation efficiency is crucial for understanding and manipulating CRISPR-Cas systems.

Purpose of the Study:

• To develop a novel system for screening and identifying CRISPR-Cas adaptation proteins with improved capabilities.
• To discover Cas1 and Cas2 protein mutants exhibiting enhanced DNA adaptation efficiency.
• To characterize the in vitro and in vivo activities of identified Cas1 mutants.

Main Methods:

• Established a robust perpetual DNA packaging and transfer (PeDPaT) system using T7 phage for efficient plasmid transduction.
• Utilized the PeDPaT system to screen for mutants with increased CRISPR-Cas adaptation efficiency.
• Performed in vitro assays to assess DNA binding, integration, and disintegration activities of mutant Cas1 proteins.

Main Results:

• Identified two Cas1 protein mutants with up to 10-fold enhanced in vivo adaptation efficiency.
• One mutant displayed increased in vitro DNA integration and binding activities.
• Another mutant exhibited enhanced in vitro DNA disintegration activity.
• Observed a decrease in protospacer adjacent motif specificity for the identified mutants.

Conclusions:

• The PeDPaT system is a powerful tool for discovering and enhancing CRISPR-Cas adaptation proteins.
• Mutant Cas1 proteins demonstrate altered DNA binding, integration, and disintegration activities, impacting adaptation efficiency.
• The developed technology holds potential for various screening applications requiring efficient DNA transduction.