CRISPR-Mediated Editing of the B Cell Receptor in Primary Human B Cells.

Authors:
Greiner V, Bou Puerto R, Liu S, Herbel C, Carmona EM, Goldberg MS.
In:
Source: iScience
Publication Date: (2019)
Issue: 12: 369-378
Research Area:
Immunotherapy / Hematology
Cells used in publication:
B cell, human
Species: human
Tissue Origin: blood
Platform:
Nucleofector™ I/II/2b
Experiment

15 µg Cas9 (~100 pmol) were incubated with 20 µg gRNA (~580 pmol) for 20 min at room temperature (RT). 100 pmol of single-stranded HR template was added right before nucleofection B cells were nucleofected with the Amaxa® Human B Cell Nucleofector® Kit (Lonza). 2-5 x106 cells per transfection were collected, washed once with PBS, and resuspended in 100 µl Nucleofector solution. The cell suspension was added to the Cas9 RNP/HR template mix, and cells were nucleofected with program V-015 in a Nucleofector™ 2b device (Lonza).

For nucleofection of Cas9 as plasmid or mRNA: 
2-5 µg lentiCas9-eGFP (Addgene, #63592) or Cas9 mRNA 5meC, ? (Trilink) weremixed with 20 µg gRNA (~580 pmol) and nucleofected under the same conditions.

Abstract

Vaccination approaches have generally focused on the antigen rather than the resultant antibodies generated, which differ greatly in quality and function between individuals. The ability to replace the variable regions of the native B cell receptor (BCR) heavy and light chain loci with defined recombined sequences of a preferred monoclonal antibody could enable curative adoptive cell transfer. We report CRISPR-mediated homologous recombination (HR) into the BCR of primary human B cells. Ribonucleoprotein delivery enabled editing at the model CXCR4 locus, as demonstrated by T7E1 assay, flow cytometry, and TIDE analysis. Insertion via HR was confirmed by sequencing, cross-boundary PCR, and restriction digest. Optimized conditions were used to achieve HR at the BCR variable heavy and light chains. Insertion was confirmed at the DNA level, and transgene expression from the native BCR promoters was observed. Reprogramming the specificity of antibodies in the genomes of B cells could have clinical importance.