Related Experiment Video

Updated: Jun 18, 2026

CIRCLE-Seq for Interrogation of Off-Target Gene Editing
08:23

CIRCLE-Seq for Interrogation of Off-Target Gene Editing

Published on: November 1, 2024

Target RNA-triggered CRISPR-Cas12a2 preferentially cleaves collateral DNA over RNA

Sobita Kunwar1, Thomson Hallmark1, Sudeshna Manna2

  • 1Department of Chemistry and Biochemistry, Utah State University, Logan, UT 84322, United States.

Nucleic Acids Research
|June 16, 2026
PubMed

Insights

Cas12a2 exhibits collateral DNA cleavage preference over RNA, even with abundant RNA. This DNA preference is key for developing Cas12a2-based technologies and understanding its immune functions.

Area of Science:

  • Molecular Biology
  • Biochemistry
  • Genetics

Background:

  • CRISPR-Cas systems utilize collateral cleavage for defense.
  • Cas12a2 is a unique RNA-guided nuclease with broad collateral cleavage activity.
  • The mechanisms and substrate preferences of Cas12a2 collateral cleavage are largely unknown.

Purpose of the Study:

  • To elucidate the biochemical mechanisms and substrate preferences of Cas12a2 collateral cleavage.
  • To investigate the role of specific residues in Cas12a2's dsDNA cleavage.
  • To explore the potential applications of Cas12a2's substrate preference in biotechnology.

Main Methods:

  • Enzyme kinetics and inhibition assays were used to determine substrate preferences.
  • Site-directed mutagenesis and enzyme kinetics were employed to study the dsDNA cleavage mechanism.
  • Plasmid cleavage assays and probe cleavage experiments were conducted to assess Cas12a2 activity.

Main Results:

  • Cas12a2 demonstrates a strong preference for cleaving collateral DNA over RNA substrates.
  • The 'aromatic clamp' residues are crucial for the dsDNA cleavage mechanism by stabilizing unwound DNA.
  • RNA-activated Cas12a2 effectively cleaves ssDNA probes in the presence of excess RNA, unlike Cas13a.

Conclusions:

  • This study provides fundamental kinetic and biochemical insights into Cas12a2's collateral cleavage mechanism and substrate specificity.
  • The findings highlight Cas12a2's preferential DNA cleavage, offering a basis for developing novel CRISPR-based technologies.
  • Understanding Cas12a2's collateral activity has direct implications for its role in microbial immunity and biotechnological applications.

More Related Videos

Selection-dependent and Independent Generation of CRISPR/Cas9-mediated Gene Knockouts in Mammalian Cells11:35

Selection-dependent and Independent Generation of CRISPR/Cas9-mediated Gene Knockouts in Mammalian Cells

Published on: June 16, 2017

Enhanced Genome Editing with Cas9 Ribonucleoprotein in Diverse Cells and Organisms09:51

Enhanced Genome Editing with Cas9 Ribonucleoprotein in Diverse Cells and Organisms

Published on: May 25, 2018

Related Concept Videos

CRISPR01:59

CRISPR

Genome editing technologies allow scientists to modify an organism’s DNA via the addition, removal, or rearrangement of genetic material at specific genomic locations. These types of techniques could potentially be used to cure genetic disorders such as hemophilia and sickle cell anemia. One popular and widely used DNA-editing research tool that could lead to safe and effective cures for genetic disorders is the CRISPR-Cas9 system. CRISPR-Cas9 stands for Clustered Regularly Interspaced Short...
CRISPR01:59

CRISPR

Genome editing technologies allow scientists to modify an organism’s DNA via the addition, removal, or rearrangement of genetic material at specific genomic locations. These types of techniques could potentially be used to cure genetic disorders such as hemophilia and sickle cell anemia. One popular and widely used DNA-editing research tool that could lead to safe and effective cures for genetic disorders is the CRISPR-Cas9 system. CRISPR-Cas9 stands for Clustered Regularly Interspaced Short...
CRISPR/Cas9 Genome Editing01:28

CRISPR/Cas9 Genome Editing

The CRISPR-Cas system serves as a bacterial defense mechanism against invading genetic elements such as viruses and plasmids, forming the foundation for its adaptation as a powerful genome-editing tool. Originally discovered in prokaryotes, this system has been repurposed to revolutionize genetic engineering across a wide range of organisms, including plants, animals, and humans. The core component, Cas9, is an endonuclease derived from Streptococcus pyogenes, capable of introducing...
CRISPR and crRNAs02:53

CRISPR and crRNAs

Bacteria and archaea are susceptible to viral infections just like eukaryotes; therefore, they have developed a unique adaptive immune system to protect themselves. Clustered regularly interspaced short palindromic repeats and CRISPR-associated proteins (CRISPR-Cas) are present in more than 45% of known bacteria and 90% of known archaea.
The CRISPR-Cas system stores a copy of foreign DNA in the host genome and uses it to identify the foreign DNA upon reinfection. CRISPR-Cas has three different...
Homologous Recombination02:31

Homologous Recombination

The basic reaction of homologous recombination (HR) involves two chromatids that contain DNA sequences sharing a significant stretch of identity. One of these sequences uses a strand from another as a template to synthesize DNA in an enzyme-catalyzed reaction. The final product is a novel amalgamation of the two substrates. To ensure an accurate recombination of sequences, HR is restricted to the S and G2 phases of the cell cycle. At these stages, the DNA has been replicated already and the...
Conservative Site-specific Recombination and Phase Variation02:53

Conservative Site-specific Recombination and Phase Variation

Because the DNA segments are cut and reorganized in a direction-specific manner, site-specific recombination has emerged as an efficient genetic engineering technique. Flippase and Cyclization recombinases or Flp and Cre, respectively, are two members of the tyrosine recombinase family derived from bacteriophages, that are used to mediate site-specific DNA insertions, deletions, and targeted expression of proteins in mammalian cell lines.
The recognition sites for Cre recombinase called LoxP...