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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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Conservative Site-specific Recombination and Phase Variation02:53

Conservative Site-specific Recombination and Phase Variation

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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...
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The Antiviral System of Bacteria and Archaea: CRISPR01:23

The Antiviral System of Bacteria and Archaea: CRISPR

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CRISPR stands for Clustered Regularly Interspaced Short Palindromic Repeats is a adaptive immune system found in bacteria and archaea that protects against viral infections. This system enables prokaryotic cells to identify, remember, and neutralize foreign genetic elements, primarily bacteriophages, by storing fragments of the invader’s DNA as a genetic memory.The CRISPR immune response begins during an initial infection. Cas (CRISPR-associated) proteins play a central role in this...
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Related Experiment Video

Updated: Oct 5, 2025

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

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Systematic decomposition of sequence determinants governing CRISPR/Cas9 specificity.

Rongjie Fu1, Wei He1, Jinzhuang Dou1

  • 1Department of Epigenetics and Molecular Carcinogenesis, The University of Texas MD Anderson Cancer Center, Smithville, TX, 78957, USA.

Nature Communications
|January 26, 2022
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Summary

New rules for CRISPR/Cas9 genome editing specificity were discovered, improving prediction of off-target effects and enabling precise allele-specific editing for cancer mutations.

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Using Sniper-Cas9 to Minimize Off-target Effects of CRISPR-Cas9 Without the Loss of On-target Activity Via Directed Evolution
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Substrate Generation for Endonucleases of CRISPR/Cas Systems
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Substrate Generation for Endonucleases of CRISPR/Cas Systems

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Using Sniper-Cas9 to Minimize Off-target Effects of CRISPR-Cas9 Without the Loss of On-target Activity Via Directed Evolution
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Substrate Generation for Endonucleases of CRISPR/Cas Systems
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Substrate Generation for Endonucleases of CRISPR/Cas Systems

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Area of Science:

  • Molecular Biology
  • Genetics
  • Bioinformatics

Background:

  • CRISPR/Cas9 genome editing relies on guide RNA (gRNA) and DNA sequence complementarity.
  • Understanding sequence-dependent rules for off-target effects is crucial for precise genome editing.

Purpose of the Study:

  • To systematically investigate sequence determinants of CRISPR/Cas9 off-target effects.
  • To develop a predictive model for Cas9-mediated off-target activity.
  • To explore allele-specific genome editing strategies.

Main Methods:

  • Developed a dual-target system to measure relative cleavage rates (off-on ratios) for 1902 gRNAs across 13,314 synthetic targets.
  • Analyzed sequence rules governing off-targeting, including guide-intrinsic mismatch tolerance (GMT) and combinatorial mismatch effects.
  • Developed the MOFF (Model Of guide-RNA-guided Fidelity) predictor.

Main Results:

  • Identified two key sequence rules for off-targeting: GMT and an "epistasis-like" combinatorial effect of mismatches.
  • The combinatorial effect is linked to R-loop formation free energy and explained by a multi-state kinetic model.
  • MOFF accurately predicts Cas9 off-target effects.
  • Demonstrated improved allele-specific editing selectivity using mismatched guides and MOFF predictions on 18 cancer hotspot mutations.

Conclusions:

  • Sequence rules governing CRISPR/Cas9 specificity have been elucidated.
  • MOFF provides a powerful tool for predicting and minimizing off-target effects.
  • The combinatorial mismatch effect enables enhanced allele-specific genome editing strategies for therapeutic applications.