Combining different CRISPR nucleases for simultaneous knock-in and base editing prevents translocations in multiplex-edited CAR T cells

Glaser V, Flugel C, Kath J, Du W, Drosdek V, Franke C, Stein M, Pruß A, Schmueck-Henneresse M, Volk HD, Reinke P, Wagner DL
Source: Genome Biology
Publication Date: (2023)
Issue: 24(1): 89
Research Area:
Immunotherapy / Hematology
Cells used in publication:
T cell, human stim.
Species: human
Tissue Origin: blood
Culture Media:
4D-Nucleofector® X-Unit

RNP formulation for gene editing: Synthetic modified gRNA (sgRNA: Cas9 or crRNA: Cas12a) sequences which were previously described [41, 42, 61] were purchased from Integrated DNA Technologies (IDT), carefully resuspended in nuclease-free 1 × TE buffer at 100 µM concentration, aliquoted and stored at - 20 °C prior to use (Additional file 2: Table S3). Per electroporation of 1–1.5 × 10^6 primary human T cells, 0.5 µL of an aqueous solution of 15- to 50-kDa poly(L-glutamic acid) (PGA) (Sigma-Aldrich, 100 µg/µL) was mixed with 0.48 µL of TRAC -specific modified sgRNA (Cas9) or TRAC -specific modified crRNA (Cas12a) by pipetting thoroughly. Then, 0.4 µL recombinant Streptococcus pyogenes Cas9 protein (Alt-R S.p. Cas9 Nuclease V3; IDT; 10 µg/µL = 61 µM) or Acidaminococcus sp. BV3L6 Cas12a (Alt-R A.s.Cas12a (Cpf1) Ultra; IDT; 10 µg/µL = 63 µM) are added and mixed by thorough pipetting. The molar ratio of Cas9/Cas12a and sgRNA was ~ 1:2. The mixture was incubated for 15 min at room temperature (RT) to allow for RNP formation and placed on ice. For KI experiments, 0.5 µL HDRT (1 µg/µL) per 10^6 cells was added at least 5 min prior transfection.

Transfection of gene editors: Primary human T cells were harvested approx. 48 h after anti-CD3/CD28 stimulation and washed twice in sterile PBS by centrifugation with 100 × g for 10 min at RT. Depending on the condition, modified mRNA (2 µg, unless stated otherwise) and/or additional sgRNA (0.48 µL each) was added to 1.88 µL of RNP/HDRT suspension. The harvested cells were resuspended in 20 µL ice-cold P3 electroporation buffer (Lonza) for electroporation of 1–1.5 × 10^6 cells. The exposure time to the electroporation buffers was kept to a minimum, and 20 µL of resuspended cells was transferred to the RNP/HDRT (+ mRNA and sgRNA) suspension, mixed thoroughly, and transferred into a 16-well electroporation strip (20 µL = 1–1.5 10^6 cells per well, Lonza). Prior to electroporation, the strips or cartridges were gently tapped onto the bench several times to ensure the placement of the liquid on the bottom of the electroporation vessel without any trapped air (bubbles). Electroporation was performed on a 4D-Nucleofector Device (Lonza) using the program EH-115. Directly after electroporation, pre-warmed T cell medium was added to the cells (90 µL per well). Afterward, the cells were carefully resuspended and transferred to 96-well round-bottom plates (50 µL/well) containing 150 µL pre-warmed T cell medium per well at a density of 0.5 × 10^6 cells per well.

T cell expansion after electroporation:  First medium change or first splitting of cells was performed 18 h after electroporation, unless stated otherwise. Cells were expanded in T cell medium on 96-well round-bottom plates or 24-well plates. T cells were split when culture-medium turned orange/yellow, indicating pH change. Typically, within the first 2 to 3 days after electroporation, T cells were split (50:50) every day or every other day. Later T cells were split, or medium was changed every 2 to 3 days. Depending on the readout, some of the T cells were pelleted and stored at - 20 °C until genomic DNA extraction. In other experiments, T cells were further expanded, counted on days 1, 4, 7, and 14 using flow cytometry to track the expansion, followed by cryopreservation in freezing medium (FCS containing 10% DMSO).


Background: Multiple genetic modifications may be required to develop potent off-the-shelf chimeric antigen receptor (CAR) T cell therapies. Conventional CRISPR-Cas
nucleases install sequence-specific DNA double-strand breaks (DSBs), enabling gene knock-out or targeted transgene knock-in. However, simultaneous DSBs provoke a high rate of genomic rearrangements which may impede the safety of the edited cells.

Results: Here, we combine a non-viral CRISPR-Cas9 nuclease-assisted knock-in and Cas9-derived base editing technology for DSB free knock-outs within a single intervention. We demonstrate efficient insertion of a CAR into the T cell receptor alpha constant (TRAC) gene, along with two knock-outs that silence major histocompatibility complexes (MHC) class I and II expression. This approach reduces translocations to 1.4% of edited cells. Small insertions and deletions at the base editing target sites indicate guide RNA exchange between the editors. This is overcome by using CRISPR enzymes of distinct evolutionary origins. Combining Cas12a Ultra for CAR knock-in and a Cas9- derived base editor enables the efficient generation of triple-edited CAR T cells with a translocation frequency comparable to unedited T cells. Resulting TCR- and MHCnegative CAR T cells resist allogeneic T cell targeting in vitro.
Conclusions: We outline a solution for non-viral CAR gene transfer and efficient gene silencing using different CRISPR enzymes for knock-in and base editing to prevent
translocations. This single-step procedure may enable safer multiplex-edited cell products and demonstrates a path towards off-the-shelf CAR therapeutics.