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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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CRISPR01:59

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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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Homologous Recombination02:31

Homologous Recombination

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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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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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Related Experiment Video

Updated: Oct 2, 2025

A Rapid and Facile Pipeline for Generating Genomic Point Mutants in C. elegans Using CRISPR/Cas9 Ribonucleoproteins
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Strategies for High-Efficiency Mutation Using the CRISPR/Cas System.

Shuying Feng1, Zilong Wang1, Aifang Li1

  • 1Medical College, Henan University of Chinese Medicine, Zhengzhou, China.

Frontiers in Cell and Developmental Biology
|February 24, 2022
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Summary

This review details strategies to enhance CRISPR-Cas gene editing efficiency and specificity. It covers methods to overcome limitations like off-target effects and improve delivery for broader applications.

Keywords:
CRISPR/Cas systemhighly efficientmutantoff-target effectoptimized strategies

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Area of Science:

  • Molecular Biology
  • Genetics
  • Biotechnology

Background:

  • CRISPR-Cas systems offer revolutionary gene-editing capabilities with high specificity and efficiency.
  • These systems hold significant potential for genetic manipulation across diverse organisms and cell types.
  • Existing limitations include off-target effects, delivery challenges, and adverse outcomes.

Purpose of the Study:

  • To comprehensively review strategies for improving CRISPR-Cas gene editing efficiency and specificity.
  • To discuss newly emerging approaches and address current challenges in CRISPR-Cas system utilization.
  • To provide practical guidance for expanding the applicability of CRISPR-Cas technology.

Main Methods:

  • Review of established strategies: reducing off-target effects, sgRNA design/modification, optimizing editing parameters (time, temperature), delivery systems, and sgRNA enrichment.
  • Detailed discussion of emerging techniques: Cas variants, anti-CRISPR proteins, and mutant enrichment.
  • In-depth analysis of current challenges and future applications.

Main Results:

  • Numerous strategies exist to enhance CRISPR-Cas gene editing efficiency and mitigate off-target effects.
  • Novel approaches like Cas variants and anti-CRISPR proteins show promise for refining the technology.
  • Optimization of delivery systems and sgRNA enrichment are crucial for successful gene editing.

Conclusions:

  • CRISPR-Cas systems require continuous improvement to overcome inherent limitations and maximize their potential.
  • This review serves as a reference for enhancing CRISPR-Cas system maturity and practical application.
  • Further research and development are essential for expanding the scope of CRISPR-Cas technology in various fields.