Engineering of Primary Human B cells with CRISPR/Cas9 Targeted Nuclease

Abstract B cells offer unique opportunities for gene therapy because of their ability to secrete large amounts of protein in the form of antibody and persist for the life of the organism as plasma cells. Here, we report optimized CRISPR/Cas9 based genome engineering of primary human B cells. Our pro...

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Autores principales:	Matthew J. Johnson, Kanut Laoharawee, Walker S. Lahr, Beau R. Webber, Branden S. Moriarity
Formato:	article
Lenguaje:	EN
Publicado:	Nature Portfolio 2018
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/0cddf17a9e6a4d6090ed118300ef3ebf
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spelling	oai:doaj.org-article:0cddf17a9e6a4d6090ed118300ef3ebf2021-12-02T15:07:44ZEngineering of Primary Human B cells with CRISPR/Cas9 Targeted Nuclease10.1038/s41598-018-30358-02045-2322https://doaj.org/article/0cddf17a9e6a4d6090ed118300ef3ebf2018-08-01T00:00:00Zhttps://doi.org/10.1038/s41598-018-30358-0https://doaj.org/toc/2045-2322Abstract B cells offer unique opportunities for gene therapy because of their ability to secrete large amounts of protein in the form of antibody and persist for the life of the organism as plasma cells. Here, we report optimized CRISPR/Cas9 based genome engineering of primary human B cells. Our procedure involves enrichment of CD19+ B cells from PBMCs followed by activation, expansion, and electroporation of CRISPR/Cas9 reagents. We are able expand total B cells in culture 10-fold and outgrow the IgD+ IgM+ CD27− naïve subset from 35% to over 80% of the culture. B cells are receptive to nucleic acid delivery via electroporation 3 days after stimulation, peaking at Day 7 post stimulation. We tested chemically modified sgRNAs and Alt-R gRNAs targeting CD19 with Cas9 mRNA or Cas9 protein. Using this system, we achieved genetic and protein knockout of CD19 at rates over 70%. Finally, we tested sgRNAs targeting the AAVS1 safe harbor site using Cas9 protein in combination with AAV6 to deliver donor template encoding a splice acceptor-EGFP cassette, which yielded site-specific integration frequencies up to 25%. The development of methods for genetically engineered B cells opens the door to a myriad of applications in basic research, antibody production, and cellular therapeutics.Matthew J. JohnsonKanut LaoharaweeWalker S. LahrBeau R. WebberBranden S. MoriarityNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 8, Iss 1, Pp 1-9 (2018)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Matthew J. Johnson Kanut Laoharawee Walker S. Lahr Beau R. Webber Branden S. Moriarity Engineering of Primary Human B cells with CRISPR/Cas9 Targeted Nuclease
description	Abstract B cells offer unique opportunities for gene therapy because of their ability to secrete large amounts of protein in the form of antibody and persist for the life of the organism as plasma cells. Here, we report optimized CRISPR/Cas9 based genome engineering of primary human B cells. Our procedure involves enrichment of CD19+ B cells from PBMCs followed by activation, expansion, and electroporation of CRISPR/Cas9 reagents. We are able expand total B cells in culture 10-fold and outgrow the IgD+ IgM+ CD27− naïve subset from 35% to over 80% of the culture. B cells are receptive to nucleic acid delivery via electroporation 3 days after stimulation, peaking at Day 7 post stimulation. We tested chemically modified sgRNAs and Alt-R gRNAs targeting CD19 with Cas9 mRNA or Cas9 protein. Using this system, we achieved genetic and protein knockout of CD19 at rates over 70%. Finally, we tested sgRNAs targeting the AAVS1 safe harbor site using Cas9 protein in combination with AAV6 to deliver donor template encoding a splice acceptor-EGFP cassette, which yielded site-specific integration frequencies up to 25%. The development of methods for genetically engineered B cells opens the door to a myriad of applications in basic research, antibody production, and cellular therapeutics.
format	article
author	Matthew J. Johnson Kanut Laoharawee Walker S. Lahr Beau R. Webber Branden S. Moriarity
author_facet	Matthew J. Johnson Kanut Laoharawee Walker S. Lahr Beau R. Webber Branden S. Moriarity
author_sort	Matthew J. Johnson
title	Engineering of Primary Human B cells with CRISPR/Cas9 Targeted Nuclease
title_short	Engineering of Primary Human B cells with CRISPR/Cas9 Targeted Nuclease
title_full	Engineering of Primary Human B cells with CRISPR/Cas9 Targeted Nuclease
title_fullStr	Engineering of Primary Human B cells with CRISPR/Cas9 Targeted Nuclease
title_full_unstemmed	Engineering of Primary Human B cells with CRISPR/Cas9 Targeted Nuclease
title_sort	engineering of primary human b cells with crispr/cas9 targeted nuclease
publisher	Nature Portfolio
publishDate	2018
url	https://doaj.org/article/0cddf17a9e6a4d6090ed118300ef3ebf
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_version_	1718388430734360576

Engineering of Primary Human B cells with CRISPR/Cas9 Targeted Nuclease

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