Related Experiment Video

Updated: Dec 14, 2025

Enhanced Genome Editing with Cas9 Ribonucleoprotein in Diverse Cells and Organisms
09:51

Enhanced Genome Editing with Cas9 Ribonucleoprotein in Diverse Cells and Organisms

Published on: May 25, 2018

35.1K

Enhancement of target specificity of CRISPR-Cas12a by using a chimeric DNA-RNA guide

Hanseop Kim1,2, Wi-Jae Lee1,3, Yeounsun Oh1,4

  • 1Futuristic Animal Resource & Research Center (FARRC), Korea Research Institute of Bioscience and Biotechnology (KRIBB), Cheongju, Korea.

Nucleic Acids Research
|July 21, 2020
PubMed

Insights

Researchers modified CRISPR-Cas12a (Cpf1) by creating a chimeric (cr)RNA to enhance genome editing specificity. This innovation reduces off-target DNA cleavage, improving safety for potential genetic disease therapies.

Area of Science:

  • Molecular Biology
  • Gene Editing Technologies
  • Biotechnology

Background:

  • CRISPR-Cas9 is a standard for targeted genome engineering.
  • CRISPR-Cas12a (Cpf1) offers distinct advantages, including recognition of thymine-rich PAM sequences and higher guide RNA mismatch sensitivity, leading to lower off-target cleavage compared to Cas9.
  • However, Cas12a's tolerance for mismatches away from the PAM site presents challenges for therapeutic applications, necessitating strategies to mitigate off-target effects.

Purpose of the Study:

  • To investigate and address off-target DNA cleavage mediated by the CRISPR-Cas12a system.
  • To develop a modified Cas12a system with improved specificity and reduced off-target activity for genome editing.
  • To propose a model for the mechanism of chimeric (cr)RNA-guided Cas12a and SpCas9 nickase in intracellular settings.

Main Methods:

  • Development of a chimeric (cr)RNA by partially substituting RNA with DNA to alter base pairing energy with target DNA.
  • Investigation of off-target cleavage patterns of the modified CRISPR-Cas12a system.
  • Modeling the intracellular action of chimeric (cr)RNA-guided CRISPR-Cas12a and SpCas9 nickase.

Main Results:

  • A novel CRISPR-Cas12a system utilizing a chimeric (cr)RNA was successfully developed.
  • The modified Cas12a system demonstrated highly specific and effective induction of mutations in target DNA sequences.
  • The chimeric guide-based CRISPR-Cas12a system exhibited reduced off-target cleavage compared to conventional Cas12a.

Conclusions:

  • The developed chimeric (cr)RNA-guided CRISPR-Cas12a system significantly reduces off-target cleavage, enhancing genome editing safety.
  • This improved specificity and safety profile make the technology a promising candidate for therapeutic applications in genetic diseases.
  • The study provides a foundation for advancing CRISPR-based gene therapies with greater precision and reduced risks.

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

13.1K
CIRCLE-Seq for Interrogation of Off-Target Gene Editing08:23

CIRCLE-Seq for Interrogation of Off-Target Gene Editing

Published on: November 1, 2024

1.2K

Related Concept Videos

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...
1.4K
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...
56.8K
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...
61.8K
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...
6.5K