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Simplified CRISPR-Mediated DNA Editing in Multicellular Eukaryotes

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Gene, Drug, and Tissue Engineering

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2575))

Abstract

The CRISPR-Cas9 system is becoming an imperative tool to edit the genome of various organisms. The gene-editing study by the CRISPR-Cas9 system has revolutionized the diverse field of biomedical research, genome engineering, and gene therapy. CRISPR-Cas9 system has been modified to induce genome editing by small-guide RNAs, which function together with Cas9 nuclease for sequence-specific cleavage of target sequences. Here, we describe the simplified protocol of CRISPR-Cas9-mediated DNA editing in multicellular eukaryotes, including the construction of gRNA plasmids into vectors, screening of positive clones, transfections into 293FT cell line, and transduction into Jurkat cells. We also describe different bioinformatic tools to design suitable gRNAs with increased efficiency and decreased off-target events. Further, we describe the assessments of DNA editing by indel mutations and sequencing in transduced cells.

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Author information

Authors and Affiliations

  1. Department of Internal Medicine, UT Southwestern Medical Center, Dallas, TX, USA

    Ritesh Kumar

  2. School of Medicine, Ophthalmology, Visual and Anatomical Sciences, Wayne State University, Detroit, MI, USA

    Kartikeya Tiwari

  3. Department of Biotechnology, National Institute of Technology, Warangal, Telangana, India

    Prakash Saudagar

Authors
  1. Ritesh Kumar
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  2. Kartikeya Tiwari
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  3. Prakash Saudagar
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Corresponding author

Correspondence to Prakash Saudagar .

Editor information

Editors and Affiliations

  1. Bioengineering Department, Imperial College London, London, UK

    Glaucia C. Pereira

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© 2023 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature

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Kumar, R., Tiwari, K., Saudagar, P. (2023). Simplified CRISPR-Mediated DNA Editing in Multicellular Eukaryotes. In: Pereira, G.C. (eds) Gene, Drug, and Tissue Engineering. Methods in Molecular Biology, vol 2575. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2716-7_11

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  • DOI: https://doi.org/10.1007/978-1-0716-2716-7_11

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-2715-0

  • Online ISBN: 978-1-0716-2716-7

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