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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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Updated: Sep 7, 2025

A Standard Methodology to Examine On-site Mutagenicity As a Function of Point Mutation Repair Catalyzed by CRISPR/Cas9 and SsODN in Human Cells
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Site-Directed Mutagenesis Method Mediated by Cas9.

Wanping Chen1, Wenwen She1, Aitao Li1

  • 1State Key Laboratory of Biocatalysis and Enzyme Engineering, Hubei Collaborative Innovation Center for Green Transformation of Bio-resources, School of Life Sciences, Hubei University, Wuhan, People's Republic of China.

Methods in Molecular Biology (Clifton, N.J.)
|June 21, 2022
PubMed
Summary
This summary is machine-generated.

This study introduces a novel CRISPR/Cas9-mediated mutagenic (ICM) system for rapid, PCR-free gene mutagenesis. The system efficiently generates designed mutants and saturation libraries, accelerating synthetic biology research.

Keywords:
CRISPR/Cas9PCR-independentSaturation mutagenesisSite-directed mutagenesisT5 exonuclease

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Area of Science:

  • Molecular Biology
  • Synthetic Biology
  • Biotechnology

Background:

  • CRISPR/Cas9 technology has revolutionized genome editing.
  • Efficient construction of mutant libraries is crucial for directed evolution and synthetic biology.
  • Current methods like PCR-based mutagenesis can be time-consuming and introduce biases.

Purpose of the Study:

  • To develop a rapid, PCR-independent in vitro CRISPR/Cas9-mediated mutagenic (ICM) system.
  • To enable efficient construction of designed mutants and site-saturation mutagenesis libraries.
  • To provide an alternative to PCR-based methods for creating high-quality mutant libraries.

Main Methods:

  • Utilized CRISPR/Cas9 with single guide RNAs for double digestion of plasmid DNA.
  • Employed T5 exonuclease to generate homologous regions on the linearized plasmid.
  • Introduced desired mutations via a short double-stranded DNA (dsDNA) insert, followed by repair in E. coli.

Main Results:

  • Demonstrated high efficiency and accuracy in generating single and multiple site-directed mutations.
  • Successfully performed mutagenesis on large-sized plasmids.
  • Validated the system as a viable alternative to PCR-based saturation mutagenesis.

Conclusions:

  • The ICM system offers a rapid and efficient method for creating designed mutants and saturation libraries.
  • This technology facilitates the generation of high-quality mutant libraries for directed evolution.
  • The ICM system holds significant potential for advancing synthetic biology research.