Potent CRISPR-Cas9 inhibitors from Staphylococcus genomes

Area of Science:

• Molecular Biology
• Genetics
• Biochemistry

Background:

• Anti-CRISPRs (Acrs) are phage-encoded proteins that inhibit CRISPR-Cas systems.
• Staphylococcus aureus Cas9 (SauCas9) is an alternative to Streptococcus pyogenes Cas9 (SpyCas9) for genome editing.
• Discovery of Acrs targeting SauCas9 is crucial for expanding genome editing toolkits.

Purpose of the Study:

• To identify novel anti-CRISPR proteins targeting Staphylococcus aureus Cas9 (SauCas9).
• To characterize the inhibitory mechanisms and functional domains of newly discovered SauCas9 inhibitors.
• To evaluate the efficacy of these inhibitors in blocking SauCas9-mediated genome editing in human cells.

Main Methods:

• Utilized self-targeting CRISPR screening and genomic search strategies to identify potential Acrs.
• Expressed and purified identified Acrs (AcrIIA13, AcrIIA14, AcrIIA15) for functional assays.
• Assessed inhibition of SauCas9 DNA cleavage activity in vitro and genome editing efficiency in human cells.
• Investigated DNA binding properties of the conserved N-terminal domain of the Acrs.

Main Results:

• Identified and characterized three potent inhibitors of SauCas9: AcrIIA13, AcrIIA14, and AcrIIA15.
• Discovered that a conserved N-terminal domain is dispensable for DNA cleavage inhibition, while divergent C termini are essential.
• Demonstrated robust inhibition of SauCas9 genome editing in human cells by AcrIIA13, and moderate inhibition by AcrIIA14 and AcrIIA15.
• Confirmed DNA binding activity of the N-terminal domain to an inverted repeat sequence in Acr promoters.

Conclusions:

• AcrIIA13, AcrIIA14, and AcrIIA15 are effective inhibitors of SauCas9.
• These Acrs exhibit bifunctional properties, inhibiting both DNA cleavage and genome editing.
• The conserved N-terminal domain's DNA binding capability suggests a regulatory role.
• These findings provide a new strategy for anti-CRISPR discovery and expand the toolbox for precise genome editing with SauCas9.