DNA target binding-induced pre-crRNA processing in type II and V CRISPR-Cas systems

Area of Science:

• Molecular Biology
• Biochemistry
• Genetics

Background:

• CRISPR RNA (crRNA) maturation involves precursor (pre)-crRNA processing in repeat and spacer regions.
• Repeat processing by Cas effectors is known for CRISPR-Cas9 and CRISPR-Cas12a, but spacer cleavage remained uncharacterized.
• No prior evidence existed for enzyme-mediated cleavage of pre-crRNA spacers in these systems.

Purpose of the Study:

• To investigate the role of DNA target binding in pre-crRNA spacer cleavage by Cas effectors.
• To characterize the distinct cleavage mechanisms of Cas12a and Cas9 on pre-crRNA spacers.
• To develop a novel DNA detection method utilizing the observed trans-cutting activity.

Main Methods:

• Biochemical assays to monitor pre-crRNA spacer cleavage by various Cas effectors (Cas12a, Cas12b, Cas12i, Cas12j, Cas9).
• Structural biology (crystallography) to elucidate conformational changes in Cas12a and Cas9 upon target binding.
• Development and validation of a one-step DNA detection assay based on trans-cleavage activity.

Main Results:

• DNA target binding triggers efficient pre-crRNA spacer cleavage by type II (Cas9) and type V (Cas12a, Cas12b, Cas12i, Cas12j) Cas effectors.
• Cas12a-mediated cleavage is activated by both single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA) targets.
• Cas9-mediated cleavage is specifically activated by ssDNA targets.
• Structural data revealed mechanisms of conformational activation in Cas12a and Cas9.
• A highly sensitive and specific one-step DNA detection method was established.

Conclusions:

• CRISPR-Cas systems possess latent pre-crRNA spacer cleavage activity upon DNA target recognition.
• Cas12a and Cas9 exhibit distinct target-dependent activation mechanisms for pre-crRNA spacer cleavage.
• The trans-cutting activity of pre-crRNA spacers provides a foundation for innovative diagnostic tools.