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Updated: May 21, 2025
A New Toolkit for Evaluating Gene Functions using Conditional Cas9 Stabilization
Published on: September 2, 2021
Purification and in vivo, cell-free, and in vitro characterization of CRISPR-Cas12a2
Friso T Schut1, Thomson Hallmark2, Oleg Dmytrenko1
1Helmholtz Institute for RNA-based Infection Research (HIRI), Helmholtz Centre for Infection Research (HZI), Würzburg, Germany.
Abstract:
The CRISPR-associated (Cas) nuclease Cas12a2 from Sulfuricurvum sp. PC08-66 (SuCas12a2) binds RNA targets with a complementary guide (g)RNA. Target RNA binding causes a major conformational rearrangement in Cas12a2 that activates a RuvC nuclease domain to collaterally cleave RNA, ssDNA and dsDNA, arresting growth and providing population-level immunity. Here, we report in vivo, cell-free, and in vitro methods to characterize the collateral cleavage activity of SuCas12a2 as well as a procedure for gRNA design. As part of the in vivo methods, we describe how to capture growth arrest through plasmid interference and induction of an SOS DNA damage response in the bacterium Escherichia coli. We further apply cell-free transcription-translation to affirm collateral cleavage activity triggered by an expressed RNA target. Finally, as part of the in vitro methods, we describe how to purify active nuclease and subsequently conduct biochemical cleavage assays. In total, the outlined methods should accelerate the exploration of SuCas12a2 and other related Cas nucleases, revealing new features of CRISPR biology and helping develop new CRISPR technologies for molecular diagnostics and other applications.
Insights
CRISPR-associated Cas12a2 nuclease targets RNA, activating collateral cleavage of RNA and DNA. This study details methods to characterize this activity, aiding CRISPR technology development.
Area of Science:
- Molecular Biology
- Biochemistry
- Genetics
Background:
- CRISPR-associated (Cas) nucleases are key components of adaptive immunity in prokaryotes.
- Cas12a2, specifically SuCas12a2, exhibits unique RNA-guided DNA and RNA collateral cleavage activity.
- Understanding Cas12a2's mechanism is crucial for developing novel CRISPR-based technologies.
Purpose of the Study:
- To establish and present comprehensive methods for characterizing SuCas12a2 collateral cleavage activity.
- To detail procedures for guide RNA design and nuclease purification.
- To facilitate further research into SuCas12a2 and related CRISPR systems.
Main Methods:
- In vivo assays in Escherichia coli to assess growth arrest via plasmid interference and SOS response.
- Cell-free transcription-translation systems to validate RNA-triggered collateral cleavage.
- In vitro purification of active SuCas12a2 and biochemical cleavage assays.
Main Results:
- Demonstrated SuCas12a2's ability to bind RNA targets and undergo conformational changes.
- Confirmed collateral cleavage of RNA, ssDNA, and dsDNA upon target binding.
- Established robust methods for gRNA design and nuclease characterization.
Conclusions:
- The developed methods accelerate the study of SuCas12a2 and related Cas nucleases.
- This research reveals new insights into CRISPR biology and Cas12a2 function.
- The findings support the advancement of CRISPR technologies for applications like molecular diagnostics.
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