DNase H Activity of Neisseria meningitidis Cas9

Area of Science:

• Molecular Biology
• Genetics
• Biochemistry

Background:

• Type II CRISPR systems utilize Cas9 nucleases for RNA-guided DNA targeting and double-stranded DNA break creation.
• Cas9 activity typically requires dual RNAs (CRISPR RNA [crRNA] and tracrRNA), protospacer adjacent motif (PAM) recognition, and crRNA-DNA complementarity.
• Cas9 orthologs, such as Neisseria meningitidis Cas9 (NmeCas9), are increasingly used in genome engineering applications.

Purpose of the Study:

• To investigate the DNA cleavage activities and substrate requirements of NmeCas9.
• To explore the complex PAM recognition patterns of NmeCas9.
• To determine the necessity of PAM and tracrRNA for NmeCas9's enzymatic functions.

Main Methods:

• Analysis of NmeCas9 DNA cleavage activities.
• Examination of substrate requirements, including single-stranded DNA (ssDNA).
• Investigation of PAM and tracrRNA independence for specific NmeCas9 activities.

Main Results:

• NmeCas9 exhibits RNA-guided cleavage of single-stranded DNA (ssDNA).
• This ssDNA cleavage activity is independent of PAM recognition and tracrRNA.
• Cleavage occurs at sites determined by the RNA-paired region, exhibiting DNase H-like activity without sequence specificity beyond guide pairing.

Conclusions:

• tracrRNA is not essential for all NmeCas9 enzymatic functions.
• NmeCas9 possesses novel RNA-guided ssDNA cleavage capabilities, expanding the known targeting activities of Cas9 endonucleases.
• These findings broaden the potential applications of NmeCas9 in genome engineering and molecular biology.