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Related Experiment Videos

A Convenient Cas9-based Conditional Knockout Strategy for Simultaneously Targeting Multiple Genes in Mouse.

Jiang Chen1, Yinan Du2, Xueyan He1

  • 1State Key Laboratory of Pharmaceutical Biotechnology and MOE Key Laboratory of Model Animal for Disease Study, Model Animal Research Center of Nanjing University, Nanjing, 210061, China.

Scientific Reports
|April 2, 2017
PubMed
Summary

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This summary is machine-generated.

This study introduces a novel conditional knockout (cKO) system using CRISPR/Cas9 for simultaneous gene deletion in mice. This faster, simpler method enables efficient conditional manipulation of multiple genes, advancing genetic research.

Area of Science:

  • Genetics
  • Molecular Biology
  • Genome Engineering

Background:

  • Conditional knockout (cKO) is crucial for studying protein function by enabling targeted gene deletion.
  • Traditional Cre-lox cKO systems require dual-allele loxP flanking, which is time-consuming, especially for multiple genes.
  • CRISPR/Cas9 technology allows simultaneous targeting of multiple genomic sites.

Purpose of the Study:

  • To develop a novel, efficient strategy for conditional knockout of multiple genes in mice.
  • To leverage CRISPR/Cas9's multiplex targeting capability with Cre-dependent Cas9 expression.
  • To overcome limitations of traditional cKO methods for complex genetic manipulations.

Main Methods:

  • Transgenic construction of loxP-stop-loxP (LSL) controlled Cas9 (LSL-Cas9).

Related Experiment Videos

  • Co-expression of LSL-Cas9 with sgRNAs targeting specific genes (e.g., EGFP, c-Maf, MafB).
  • Utilizing Cre recombinase to activate Cas9 expression for gene deletion.

    • Main Results:

    • Demonstrated Cre-dependent elimination of EGFP expression, validating the system.
    • Successfully achieved simultaneous deletion of c-Maf and MafB genes in mouse macrophages.
    • Showcased the system's efficacy in conditional manipulation of multiple target genes.

    Conclusions:

    • The proposed sgRNAs-LSL-Cas9 cKO system offers a simpler and faster alternative to traditional Cre-flox cKO.
    • This novel strategy facilitates feasible conditional manipulation of multiple genes.
    • The system enhances the efficiency of genetic research requiring simultaneous gene targeting.