Genome Engineering of Primary Human B Cells Using CRISPR/Cas9

Kanut Laoharawee, Matthew J. Johnson, Walker S. Lahr, Joseph J. Peterson, Beau R. Webber, Branden S. Moriarity

Research output: Contribution to journalArticlepeer-review

Abstract

B cells are lymphocytes derived from hematopoietic stem cells and are a key component of the humoral arm of the adaptive immune system. They make attractive candidates for cell-based therapies because of their ease of isolation from peripheral blood, their ability to expand in vitro, and their longevity in vivo. Additionally, their normal biological function-to produce large amounts of antibodies-can be utilized to express very large amounts of a therapeutic protein, such as a recombinant antibody to fight infection, or an enzyme for the treatment of enzymopathies. Here, we provide detailed methods for isolating primary human B cells from peripheral blood mononuclear cells (PBMCs) and activating/expanding isolated B cells in vitro. We then demonstrate the steps involved in using the CRISPR/Cas9 system for site-specific KO of endogenous genes in B cells. This method allows for efficient KO of various genes, which can be used to study the biological functions of genes of interest. We then demonstrate the steps for using the CRISPR/Cas9 system together with a recombinant, adeno-associated, viral (rAAV) vector for efficient site-specific integration of a transgene expression cassette in B cells. Together, this protocol provides a step-by-step engineering platform that can be used in primary human B cells to study biological functions of genes as well as for the development of B-cell therapeutics.

Original languageEnglish (US)
JournalJournal of visualized experiments : JoVE
Issue number165
DOIs
StatePublished - Nov 3 2020

PubMed: MeSH publication types

  • Journal Article
  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Video-Audio Media

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