Challenges of CRISPR-Based Gene Editing in Primary T Cells

Abstract

Adaptive T-cell immunotherapy holds great promise for the successful treatment of leukemia, as well as other types of cancers. More recently, it was also shown to be an effective treatment option for chronic virus infections in immunosuppressed patients. Autologous or allogeneic T cells used for immunotherapy are usually genetically modified to express novel T-cell or chimeric antigen receptors. The production of such cells was significantly simplified with the CRISPR/Cas system, allowing for the deletion or insertion of novel genes at specific locations within the genome. In this review, we describe recent methodological breakthroughs that were important for the conduction of these genetic modifications, summarize crucial points to be considered when conducting such experiments, and highlight the potential pitfalls of these approaches.

Keywords: CAR T cells; CRISPR/Cas9; T cells; adoptive T-cell therapy; gene modifications.

Figure 2

Efficacy of CRISPR/Cas9-induced modifications in T cells is affected by gene expression level. (A) CD4+ T cells and MACS-sorted from mice spleen and lymph nodes, were either immediately nucleofected before activation (protocol NAA) or were first activated and then nucleofected (protocol ANA). T-cell activation on anti-CD3/-CD28 coated wells, nucleofection with one two anti-Cxcr3 crRNAs (A: 5′-TGACTCCCCGCCCTGCCCAC-3′; B: 5′-GCTGTTCTGCTGGTCTCCAG-3′) or with a negative control crRNA (Neg Ctrl: 5′-CGTTAATCGCGTATAATACG-3′) coupled with tracrRNA and Cas9 (all components from Integrated DNA Technologies Inc.) into CRISPR/Cas9 ribonucleoproteins (RNPs), and expansion in medium supplemented with 100 U/mL of interleukin-2 was performed according to our previously published protocol [99]. (B) Representative histogram plots showing CXCR3 expression on CD3⁺CD4⁺ T cells immediately after isolation (day 0), after anti-CD3/-CD28 activation (day 2), or after additional 3 days of expansion (day 5). (C) Representative histogram plots showing CXCR3 expression on CD3⁺CD4⁺ T cells nucleofected with indicated Cas9 RNPs. (D) Gene-editing efficacy expressed as a relative decrease of CD3⁺CD4⁺CXCR3+ cells after nucleofection anti-Cxcr3 crRNAs compared to negative control group. Data are from two independent experiments (dots), and group means are shown as lines.

Figure 1

Graphical overview of steps included in the CRISPR/Cas9 gene editing in primary T cells, including (1) T-cell isolation and activation; (2) in vitro assembly of CRISPR/Cas cargo; (3) its delivery to the T cells; (4) induction of double-strand breaks that can be (5) repaired mainly by NHEJ or HDR; and (6) expansion and selection of modified cells and (7) confirmation of introduced changes by DNA sequencing and/or protein expression. Abbreviations: Cas9—CRISPR-associated protein 9; crRNA—CRISPR RNA; HDR—homology-directed repair; NHEJ—non-homologous end joining; tracrRNA—trans-activating CRISPR RNA. Figure created with BioRender.

Figure 3

CRISPR/Cas9-mediated correction of frameshift mutations via NHEJ or HDR pathway. To repair a single base-pair deletion in beta-2-microglobulin gene (B2m) (NC_000068.8:121981364), cells were nucleofected with CRISPR/Cas9 ribonucleoparticles (RNPs) containing 70 pmol of Cas9 complexed with 210 pmol of crRNA (5′-GCGTGAGTATACTTGAATTG-3′):tracrRNA complexes together with 70 pmol of electroporation enhancer or with the same RNPs in the presence of 100 pmol of single-stranded DNA HDR template (5′-GTTTTCATCTGTCTTCCCCTGTGGCCCTCAGAAACCCCTCAAATTCAAGTATACTCACGCCACCCACCGGAGAATGGGAAGCC-3′; all components from Integrated DNA Technologies Inc.). Nucleofection with prepared RNPs with or without HDR template in a total volume of 10 µL was performed by using SF cell line 4D-NucleofectorTM X Kit L and a 4D nucleofector X and Core units (all Lonza), using program for RAW 264.7 cell (pulse code: DS136). After nucleofection, cells were expanded for 2 days before they were used for (A) FACS analysis of major histocompatibility complex class I (MHC-I) re-expression of the cell surface; or (B) DNA extraction to amplify by PCR the region of interest (Forward primer 5′-GACACTGCTAAAAGCCAGGT-3′, reverse primer 5′-CAGATGGAGCGTCCAGAAAGT-3′; 98 °C 30″, 35 cycles (98 °C 5″, 55 °C 10″, 72 °C 40″), 72 °C 2′, using high fidelity DNA polymerase), sequencing it by Sanger sequencing and analyzing the results by using ICE (Synthego) to determine the type of genetic changes induced by NHEJ or HDR.