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Related Concept Videos

CRISPR/Cas9 Genome Editing01:28

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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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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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Updated: Mar 14, 2026

Genome Editing in Mammalian Cell Lines using CRISPR-Cas
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Optimization Strategies for the CRISPR-Cas9 Genome-Editing System.

Charles E Vejnar1, Miguel A Moreno-Mateos1, Daniel Cifuentes1

  • 1Department of Genetics, Yale University School of Medicine, New Haven, Connecticut 06510.

Cold Spring Harbor Protocols
|October 5, 2016
PubMed
Summary

CRISPR-Cas9 gene editing relies on active single guide RNAs (sgRNAs) for precise DNA targeting. Our CRISPRscan tool predicts sgRNA activity, enhancing genome engineering efficiency in vivo.

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Area of Science:

  • Molecular Biology
  • Genetics
  • Biotechnology

Background:

  • The CRISPR-Cas9 system, originally found in bacteria, is a revolutionary genome-editing tool for eukaryotic cells.
  • This system utilizes a Cas9 endonuclease guided by a single guide RNA (sgRNA) to target specific DNA sequences.
  • Optimizing sgRNA activity is crucial for efficient gene targeting and successful genome engineering.

Purpose of the Study:

  • To highlight recent advancements in understanding sequence features that enhance sgRNA activity.
  • To introduce CRISPRscan, a novel web tool designed to predict sgRNA targeting efficiency.
  • To demonstrate how CRISPRscan can improve the overall efficiency of CRISPR-Cas9 for in vivo genome engineering.

Main Methods:

  • Review of recent literature on CRISPR-Cas9 targeting and sgRNA activity.
  • Development and validation of the CRISPRscan web tool for predicting sgRNA efficacy.
  • Application of CRISPRscan to assess and enhance sgRNA performance in genome engineering experiments.

Main Results:

  • Significant progress has been made in identifying sequence determinants of high sgRNA activity.
  • The CRISPRscan tool effectively predicts the targeting activity of sgRNAs.
  • Utilizing CRISPRscan leads to improved efficiency in CRISPR-Cas9-mediated in vivo genome engineering.

Conclusions:

  • Understanding sgRNA sequence features is key to maximizing CRISPR-Cas9 system performance.
  • CRISPRscan provides a valuable resource for researchers to select highly active sgRNAs.
  • This tool has the potential to significantly advance the application of CRISPR-Cas9 for in vivo genome engineering.