Inhibition of CRISPR-Cas9 with Bacteriophage Proteins

Area of Science:

• Microbiology
• Molecular Biology
• Genetics

Background:

• Bacterial CRISPR-Cas systems provide adaptive immunity against phages using RNA-guided nucleases.
• Phages have evolved anti-CRISPR proteins to inhibit Class 1 CRISPR-Cas systems.
• No natural inhibitors were known for the widely used Class 2 CRISPR-Cas9 system.

Purpose of the Study:

• To identify proteins that inhibit the CRISPR-Cas9 system.
• To investigate natural mechanisms of CRISPR-Cas9 inactivation.

Main Methods:

• Bioinformatic search of cas9-containing bacterial genomes for co-existing CRISPR spacers and targets.
• Identification and characterization of potential inhibitor proteins from Listeria monocytogenes prophages.
• Functional assays in Escherichia coli and human cells to confirm inhibitory activity.

Main Results:

• Discovery of four unique type II-A CRISPR-Cas9 inhibitor proteins encoded by Listeria monocytogenes prophages.
• Over 50% of Listeria monocytogenes strains with cas9 harbor at least one prophage-encoded inhibitor.
• Two identified inhibitors effectively blocked Streptococcus pyogenes Cas9 activity in bacterial and human cells.

Conclusions:

• Natural CRISPR-Cas9 inhibitors, termed anti-CRISPRs, exist and are widespread in Listeria monocytogenes prophages.
• These anti-CRISPR proteins represent a novel class of Cas9 inhibitors.
• The discovered anti-CRISPRs can serve as tools for regulating CRISPR-Cas9 genome editing applications.