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Related Concept Videos

CRISPR01:59

CRISPR

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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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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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CRISPR and crRNAs02:53

CRISPR and crRNAs

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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.
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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CIRCLE-Seq for Interrogation of Off-Target Gene Editing
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CIRCLE-Seq for Interrogation of Off-Target Gene Editing

Published on: November 1, 2024

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CRISPR Off-Target Analysis Platforms.

Christine L Xu1, Merry Zhechao Ruan2, Sara D Ragi3

  • 1Stanford University School of Medicine, Stanford, CA, USA.

Methods in Molecular Biology (Clifton, N.J.)
|December 9, 2022
PubMed
Summary
This summary is machine-generated.

The CRISPR-Cas9 gene editing tool can cause unintended DNA breaks. This study reviews methods like GUIDE-Seq and BLESS for detecting these critical off-target effects.

Keywords:
BLESSCRISPR/Cas9Digenome-seqGUIDE-SeqHTGTSOff-target effects

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Area of Science:

  • Molecular Biology
  • Genetics
  • Bioengineering

Background:

  • The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9 system offers programmable genome and epigenome editing capabilities.
  • CRISPR/Cas9 utilizes a guide RNA to direct Cas9 endonuclease to a specific DNA target, creating double-strand breaks (DSBs).
  • A significant limitation of CRISPR/Cas9 is the potential for unintended, off-target DSBs, leading to genomic instability.

Purpose of the Study:

  • To review current CRISPR technologies and applications.
  • To highlight the critical need for sensitive assays to detect and analyze CRISPR/Cas9 off-target editing.
  • To present an overview of existing analysis platforms for identifying off-target effects.

Main Methods:

  • Review of CRISPR/Cas9 technology and its applications in genome editing.
  • Discussion of the challenges posed by off-target double-strand breaks (DSBs).
  • Description of key analytical platforms for detecting off-target effects: GUIDE-Seq, HTGTS, BLESS, and Digenome-seq.

Main Results:

  • CRISPR/Cas9 technology enables precise genome editing but carries the risk of off-target mutations.
  • Several advanced sequencing-based methods have been developed to identify these off-target DSBs.
  • These methods include genome-wide, unbiased identification of DSBs enabled by sequencing (GUIDE-Seq), high-throughput genomic translocation sequencing (HTGTS), breaks labeling, enrichments on streptavidin and next-generation sequencing (BLESS), and in vitro nuclease-digested genome sequencing (Digenome-seq).

Conclusions:

  • Accurate detection of CRISPR/Cas9 off-target effects is crucial for safe and effective gene editing applications.
  • The reviewed platforms offer sensitive and specific detection of unintended DNA modifications.
  • Continued development and application of these assays are essential for advancing CRISPR-based therapeutics and research.