Genetic engineering in primary human B cells with CRISPR-Cas9 ribonucleoproteins

J Immunol Methods. 2018 Jun;457:33-40. doi: 10.1016/j.jim.2018.03.009. Epub 2018 Mar 31.

Abstract

Genome editing in human cells with targeted nucleases now enables diverse experimental and therapeutic genome engineering applications, but extension to primary human B cells remains limited. Here we report a method for targeted genetic engineering in primary human B cells, utilizing electroporation of CRISPR-Cas9 ribonucleoproteins (RNPs) to introduce gene knockout mutations at protein-coding loci with high efficiencies that in some cases exceeded 80%. Further, we demonstrate knock-in editing of targeted nucleotides with efficiency exceeding 10% through co-delivery of oligonucleotide templates for homology directed repair. We delivered Cas9 RNPs in two distinct in vitro culture systems to achieve editing in both undifferentiated B cells and activated B cells undergoing differentiation, reflecting utility in diverse experimental conditions. In summary, we demonstrate a powerful and scalable research tool for functional genetic studies of human B cell biology that may have further applications in engineered B cell therapeutics.

Keywords: CRISPR-Cas9; Cas9 ribonucleoprotein; Genome engineering; Primary human B cells.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adolescent
  • Adult
  • B-Lymphocytes / cytology*
  • B-Lymphocytes / immunology
  • CRISPR-Cas Systems*
  • Cell Line
  • Gene Knockout Techniques
  • Genetic Engineering*
  • Humans
  • Mutation
  • Palatine Tonsil / cytology
  • Recombinational DNA Repair
  • Ribonucleoproteins / genetics*
  • Sialic Acid Binding Ig-like Lectin 2 / genetics
  • Young Adult

Substances

  • Ribonucleoproteins
  • Sialic Acid Binding Ig-like Lectin 2