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Design and Assembly of CRISPR/Cas9 Lentiviral and rAAV Vectors for Targeted Genome Editing

  • Ivette M. SandovalEmail author
  • Timothy J. Collier
  • Fredric P. Manfredsson
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1937)

Abstract

Clustered regularly interspaced short palindromic repeat (CRISPR/Cas) system has emerged as an extremely useful tool for biological research and as a potential technology for gene therapy approaches. CRISPR/Cas mediated genome editing can be used to easily and efficiently modify endogenous genes in a large variety of cells and organisms. Furthermore, a modified version of the Cas9 nuclease has been developed that can be used for regulation of endogenous gene expression and labeling of genomic loci, among other applications. This chapter provides an introduction to the basis of the technology and a detail protocol for the most classic application: gene inactivation by CRISPR/Cas9 nuclease system from Streptococcus pyogenes. This workflow can be easily adapted for other CRISPR systems and applications.

Key words

Gene editing Gene inactivation CRISPR/Cas9 Cas9 nuclease LV AAV guideRNA 

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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Ivette M. Sandoval
    • 1
    • 2
    Email author
  • Timothy J. Collier
    • 1
    • 2
  • Fredric P. Manfredsson
    • 1
    • 2
  1. 1.Department of Translational Science & Molecular Medicine, College of Human MedicineMichigan State UniversityGrand RapidsUSA
  2. 2.Mercy Health Saint Mary’sGrand RapidsUSA

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