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CRISPR Guide RNA Design pp 37–48Cite as

Chemical Modification of Guide RNAs for Improved CRISPR Activity in CD34+ Human Hematopoietic Stem and Progenitor Cells

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Part of the book series: Methods in Molecular Biology ((MIMB,volume 2162))

Abstract

Human CD34+ hematopoietic stem and progenitor cells (HSPCs) have the unique ability to repopulate the entire hematopoietic system and thus are at the center of diverse, therapeutically relevant studies. The recent development of the CRISPR/Cas9 tool made the powerful research technique of genome editing highly accessible. Our previous studies demonstrated that high editing efficiency is reached when the CRISPR/Cas9 is introduced to CD34+ HSPCs as a ribonucleoprotein (RNP) complex with chemically modified guide RNAs (gRNAs). The current protocol details a quick 4-day procedure for ex vivo genome editing in CD34+ HSPCs by RNP complexes that are targeted to a specific locus by either a single gRNA (sgRNA) or a 2-part gRNA. The delivery of RNP complexes is performed by electroporation in the presence of a nonspecific, ssDNA electroporation enhancer, which highly improves editing efficiency under the described conditions. This approach is simple and effective with the potential to accelerate many biotechnological and therapeutic applications of the CRISPR/Cas9 technology.

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Authors and Affiliations

  1. The Mina and Everard Goodman Faculty of Life Sciences, Advanced Materials and Nanotechnology Institute, Bar-Ilan University, Ramat-Gan, Israel

    Jenny Shapiro, Adi Tovin, Ortal Iancu, Daniel Allen & Ayal Hendel

Authors
  1. Jenny Shapiro
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  2. Adi Tovin
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  3. Ortal Iancu
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  4. Daniel Allen
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  5. Ayal Hendel
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Corresponding author

Correspondence to Ayal Hendel .

Editor information

Editors and Affiliations

  1. Radcliffe Department of Medicine, Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, UK

    Tudor A. Fulga

  2. Radcliffe Department of Medicine, Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, UK

    David J. H. F. Knapp

  3. Radcliffe Department of Medicine, Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, UK

    Quentin R. V. Ferry

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Shapiro, J., Tovin, A., Iancu, O., Allen, D., Hendel, A. (2021). Chemical Modification of Guide RNAs for Improved CRISPR Activity in CD34+ Human Hematopoietic Stem and Progenitor Cells. In: Fulga, T.A., Knapp, D.J.H.F., Ferry, Q.R.V. (eds) CRISPR Guide RNA Design. Methods in Molecular Biology, vol 2162. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0687-2_3

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  • DOI: https://doi.org/10.1007/978-1-0716-0687-2_3

  • Published:

  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-0686-5

  • Online ISBN: 978-1-0716-0687-2

  • eBook Packages: Springer Protocols

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