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Substrate Generation for Endonucleases of CRISPR/Cas Systems
Published on: September 8, 2012
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Functionally diverse type V CRISPR-Cas systems
Winston X Yan1, Pratyusha Hunnewell1, Lauren E Alfonse1
1Arbor Biotechnologies, Cambridge, MA 02139, USA.
Science (New York, N.Y.)
|December 8, 2018
Summary
Researchers explored novel Type V CRISPR-Cas systems, identifying Cas12c, Cas12g, Cas12h, and Cas12i. These systems show diverse DNA and RNA targeting capabilities, expanding the CRISPR toolbox for molecular biology applications.
Area of Science:
- Molecular Biology
- Genetics
- Biochemistry
Background:
- Type V CRISPR-Cas systems utilize a single effector protein, Cas12, for RNA-guided DNA targeting.
- While Cas12a and Cas12b are well-characterized, other Cas12 subtypes remain unexplored, hinting at functional diversity.
- Distinct domain architectures and RuvC sequences in uncharacterized Cas12 proteins suggest novel activities.
Purpose of the Study:
- To identify and characterize novel Cas12 proteins from Type V CRISPR-Cas systems.
- To investigate the functional diversity and enzymatic activities of newly identified Cas12 subtypes.
- To expand the repertoire of CRISPR-Cas tools for genome editing and molecular applications.
Main Methods:
- Bioinformatic identification of uncharacterized Cas12 proteins.
- Biochemical characterization of Cas12c, Cas12g, Cas12h, and Cas12i.
- Assays to determine RNA-guided DNA interference, RNase, and DNase activities.
Main Results:
- Cas12c, Cas12h, and Cas12i exhibit RNA-guided double-stranded DNA (dsDNA) interference.
- Cas12i demonstrates preferential cleavage of the complementary strand, leading to dsDNA nicking.
- Cas12g functions as an RNA-guided ribonuclease (RNase) with collateral RNase and single-strand DNase activities.
Conclusions:
- The study reveals significant functional diversity within Type V CRISPR-Cas systems, driven by distinct evolutionary paths.
- Newly characterized Cas12 proteins expand the capabilities of CRISPR technology.
- These findings provide novel tools for diverse applications in molecular biology and biotechnology.
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