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

Abstract

Summary 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.

Towards Physiologically and Tightly Regulated Vectored Antibody Therapies

“…Yet, directly targeting the variable or joining regions remains feasible. Indeed, two groups have shown efficient cleavage in these regions by designing sgRNAs that can bind to frequently kept V and J regions [86] or at the extremities (first V and last J domains, Figure 2) [87]. Several DNA templates have been created for homologous recombination to introduce either a new variable heavy region alone or with a new variable light region after cleavage ( Figure 2) [85,87].…”

“…Several DNA templates have been created for homologous recombination to introduce either a new variable heavy region alone or with a new variable light region after cleavage ( Figure 2) [85,87]. In addition, Greiner et al introduced a full-length antibody chains (heavy and light) including the constant domains ( Figure 2) [86]. The advantage here is that the antibody effector functions, carried on the constant domains, can be enhanced by mutating these regions before insertion.…”

“…The delivery methods for the Cas9 protein and the gRNA are also challenging. Retroviral vectors [88], gRNA precomplexed to Cas9 protein [85,86,89], or plasmids encoding Cas9 and gRNA can be used [87]. Alternatively, combo systems, such as the "nanoblade" platform, may represent suitable alternatives to deliver the DNA donor template, the Cas9 protein and the sgRNA [91].…”

“…The two types of light chains, κ and λ, are respectively located on chromosome 2 and 22 and comprises variable, joining and constant regions. The cutting sites targeted by different groups to edit genetically antibody loci are indicated by arrows (red [88], green [87], yellow [86], blue [85]). (b) Antibodies after editing.…”

Highly efficient and specific genome editing in human cells with paired CRISPR-Cas9 nickase ribonucleoproteins

“…In order to improve upon plasmid-based limitations, complexes comprised of purified recombinant Cas9 protein and guide RNA (RNP) have been developed for direct delivery to a multitude of different human cell types. These RNPs have been shown to cut genomic DNA rapidly after delivery and degrade rapidly, thereby decreasing off-target concerns associated with the stronger Cas9-gRNA expression characteristic of plasmid-based systems [15][16][17][18][19] .…”