Strategies for CRISPR-based knock-ins in primary human B cells and lymphoma cell lines

Abstract

Since its advent about ten years ago, the CRISPR-Cas9 system has been frequently used in biomedical applications. It has advanced various fields, and CRISPR-Cas9-based therapeutics have shown promising results in the treatment of specific hematological diseases. Furthermore, CRISPR gene editing technologies have revolutionized cancer research by enabling a broad range of genetic perturbations, including genetic knockouts and precise single nucleotide changes. This perspective focuses on the state-of-the-art methodology of CRISPR knock-ins to engineer immune cells. Since this technique relies on homology-directed repair (HDR) of double-strand breaks (DSBs) induced by the Cas9 enzyme, it can be used to introduce specific mutations into the target genome. Therefore, this methodology offers a valuable opportunity to functionally study specific mutations and to uncover their impacts not only on overall cell functions but also on the mechanisms behind cancer-related alterations in common signaling pathways. This article highlights CRISPR knock-in strategies, protocols, and applications in cancer and immune research, with a focus on diffuse large B cell lymphoma.

Keywords: DLBCL; NF-kappa B (NF-KB); gene editing (CRISPR/Cas9); knock-in; lymphoma.

Figure 1

Schematic overview of the CRISPR knock-in protocol. The Cas9 protein, sgRNA and HDR template enter the cell via electroporation, where the HDR template is replacing the original DNA sequence through homology directed repair (HDR). After successful electroporation, single cells are plated via limiting dilution and Sanger sequenced to determine the clones with a homozygous knock-in.

Figure 2

BCL10 mutations. (A) Sanger sequencing result of a BCL10 mutation. The cytosine at position 136 is replaced by a guanine, resulting in the S136X mutation. (B) Mean nuclear NF-κB (p50) translocation score assessed by ImageStream flow cytometry in wildtype (WT) or BCL10 S136X mutant TMD8 cells treated for 16 hours with DMSO or acalabrutinib. *, P ≤ 0.05, ****, P ≤ 0.0001 (one-way ANOVA). Data from 5 independent replicates. Error bars represent SEM.