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Related Concept Videos

CRISPR/Cas9 Genome Editing01:28

CRISPR/Cas9 Genome Editing

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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...
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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...
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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...
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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.
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Related Experiment Video

Updated: Nov 15, 2025

CIRCLE-Seq for Interrogation of Off-Target Gene Editing
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Improved HTGTS for CRISPR/Cas9 off-target detection.

Jianhang Yin1,2, Mengzhu Liu1, Yang Liu1

  • 1The MOE Key Laboratory of Cell Proliferation and Differentiation, Genome Editing Research Center, School of Life Sciences, Peking University, Beijing, 100871, China.

Bio-Protocol
|March 3, 2021
PubMed
Summary

We developed an improved high-throughput genome-wide translocation sequencing (iHTGTS) method to detect CRISPR-Cas9 off-target effects. This cost-effective and highly sensitive technique enhances genome editing safety assessments.

Keywords:
CRISPR-Cas9Chromosomal translocationIHTGTSLAM-HTGTSOff-target activity

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Area of Science:

  • Molecular Biology
  • Genomics
  • Biotechnology

Background:

  • CRISPR-Cas9 genome editing requires precise off-target activity evaluation for research and clinical use.
  • Linear amplification-mediated high-throughput genome-wide translocation sequencing (LAM-HTGTS) is a key method for assessing Cas9 off-target effects.
  • LAM-HTGTS relies on chromosomal translocations to capture off-target DNA double-stranded breaks (DSBs).

Purpose of the Study:

  • To introduce an improved high-throughput genome-wide translocation sequencing (iHTGTS) method.
  • To enhance the efficiency and sensitivity of detecting CRISPR-Cas9 off-target mutations.
  • To provide a more cost-effective alternative for genome-wide off-target analysis.

Main Methods:

  • Implementation of size-selection beads to improve reaction efficiency.
  • Design of a new primer system compatible with Illumina Hiseq sequencing.
  • Application of the iHTGTS method in HEK293T, K562, U2OS, and HCT116 cell lines.

Main Results:

  • The iHTGTS method demonstrated increased reaction efficiency compared to LAM-HTGTS.
  • iHTGTS exhibited significantly higher sensitivity in detecting off-target mutations.
  • The method proved effective across multiple human cell lines, including HEK293T, K562, U2OS, and HCT116.
  • iHTGTS offers a lower cost for off-target detection.

Conclusions:

  • iHTGTS is a powerful and sensitive tool for comprehensive Cas9 off-target effect assessment.
  • The improved method offers a cost-effective solution for genome editing safety evaluations.
  • iHTGTS facilitates more accurate and efficient detection of unintended genomic alterations.