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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...
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...
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...
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...
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...

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Related Experiment Video

Updated: Jun 19, 2026

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

Guide RNA reprogramming facilitates minimized tracrRNA-dependent off-target and versatile CRISPR/Cas9 engineering.

Wenxia Yu1,2, Jun Chen3, Junfan Guo4

  • 1Department of Plastic and Reconstructive Surgery, Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine; Shanghai Institute of Precision Medicine, Shanghai, China.

Nature Communications
|June 17, 2026
PubMed
Summary

Researchers found that guide RNAs (gRNAs) can be reprogrammed, leading to tracrRNA-dependent off-target effects. They developed new CRISPR-Cas9 systems to minimize these effects and enhance safety for gene editing applications.

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Related Experiment Videos

Last Updated: Jun 19, 2026

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

A New Toolkit for Evaluating Gene Functions using Conditional Cas9 Stabilization
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A New Toolkit for Evaluating Gene Functions using Conditional Cas9 Stabilization

Published on: September 2, 2021

Selection-dependent and Independent Generation of CRISPR/Cas9-mediated Gene Knockouts in Mammalian Cells
11:35

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

Published on: June 16, 2017

Area of Science:

  • Molecular Biology
  • Gene Editing Technologies
  • Biotechnology

Background:

  • Current CRISPR-Cas9 systems exhibit safety concerns, including sequence-independent off-targeting.
  • The guide RNA (gRNA) structure, specifically the crRNA:tracrRNA duplex, is splittable and reprogrammable.
  • Endogenous RNAs can hijack gRNAs, causing tracrRNA-dependent off-target (TDO) effects.

Purpose of the Study:

  • To address safety concerns and practical limitations in CRISPR-Cas9 systems.
  • To investigate and mitigate tracrRNA-dependent off-target effects.
  • To develop enhanced CRISPR platforms for improved safety and expanded applications.

Main Methods:

  • Machine learning models trained on gRNA variant screens to establish optimal gRNA design rules.
  • Engineering of crRNA variants mismatched to human/mouse transcriptomes to minimize TDO.
  • Development of reprogrammable tracrRNAs for CRISPR activation (CRISPRa) and redesigned scaffolds to prevent PAM-less Cas9 self-editing.

Main Results:

  • Identification of a novel off-target mechanism mediated by endogenous RNA hijacking of gRNAs.
  • Successful engineering of crRNA variants that significantly reduce TDO effects.
  • Creation of a separately expressed gRNA (segRNA) platform enabling multiplexed gene editing and functional annotation.

Conclusions:

  • The splittability and reprogrammability of gRNAs present a previously unrecognized off-target risk.
  • Developed strategies effectively minimize TDO, enhancing CRISPR system safety.
  • The novel segRNA platform and reprogrammable tracrRNAs offer versatile tools for advanced gene editing and cellular research.