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CRISPR/Cas9 Genome Editing01:28

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Genome Editing in Mammalian Cell Lines using CRISPR-Cas
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Genome Editing in Mammalian Cell Lines using CRISPR-Cas

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Genome engineering using CRISPR-Cas9 system.

Le Cong1, Feng Zhang

  • 1Broad Institute of MIT and Harvard, 7 Cambridge Center, Cambridge, MA, 02142, USA, congle.thu@gmail.com.

Methods in Molecular Biology (Clifton, N.J.)
|November 20, 2014
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Summary
This summary is machine-generated.

This chapter details using the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9 system for genome engineering in mammalian cells. Protocols cover site selection to detecting genomic modifications, accelerating genetic research.

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Area of Science:

  • Molecular Biology
  • Genetics
  • Biotechnology

Background:

  • The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9 system is a microbial adaptive immune mechanism.
  • Engineered CRISPR-Cas9 offers a rapid, cost-effective, and scalable method for genomic manipulation in eukaryotic cells.

Purpose of the Study:

  • To provide detailed protocols for RNA-guided genome engineering in mammalian cells using the CRISPR-Cas9 system from Streptococcus pyogenes.
  • To equip researchers with methods for efficient gene editing applications.

Main Methods:

  • Site selection and molecular cloning for CRISPR-Cas9 components.
  • Delivery of guide RNAs (gRNAs) and Cas9 nuclease into mammalian cells.
  • Verification of target DNA cleavage and detection of genomic modifications (indels, homologous recombination).

Main Results:

  • Demonstrated successful application of CRISPR-Cas9 for RNA-guided genome engineering in mammalian systems.
  • Provided comprehensive assays for validating target cleavage and genomic alterations.
  • Facilitated acceleration of both forward and reverse genetics studies.

Conclusions:

  • The described protocols enable robust and versatile genome engineering in mammalian cells.
  • The CRISPR-Cas9 system serves as a powerful tool for advancing genetic research and functional genomics.
  • These methods enhance the efficiency and scalability of genetic modification studies.