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

• Published in: Scientific reports 2018-08, Vol.8 (1), p.12144-9, Article 12144
• Main Authors: Johnson, Matthew J., Laoharawee, Kanut, Lahr, Walker S., Webber, Beau R., Moriarity, Branden S.
• Format: Article
• Language: English
• Subjects: 13/106; 13/31; 38/109; 38/44; 42/41; 631/208; 631/250; 631/61/201/2110; Antigens, CD19 - genetics; Antigens, CD19 - metabolism; B-Lymphocytes - metabolism; CD19 antigen; CD27 antigen; Cell activation; Cell culture; Cell Engineering - methods; Cells, Cultured; CRISPR; CRISPR-Cas Systems - genetics; Electroporation; Endonucleases - genetics; Gene Knockout Techniques - methods; Gene therapy; Genetic engineering; Genetic Engineering - methods; Genome editing; Genomes; Healthy Volunteers; Humanities and Social Sciences; Humans; Immunoglobulin D; Immunoglobulin M; Lymphocyte receptors; Lymphocytes B; mRNA; multidisciplinary; Nuclease; Nucleic acids; Plasma cells; Primary Cell Culture; Proteins; Reagents; RNA, Guide, CRISPR-Cas Systems - genetics; Science; Science (multidisciplinary)
• ISSN: 2045-2322; 2045-2322
• DOI: 10.1038/s41598-018-30358-0

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.