The CRISPR-Cas12a Platform for Accurate Genome Editing, Gene Disruption, and Efficient Transgene Integration in Human Immune Cells

Mohr M, Damas N, Gudmand-Høyer J, Zeeberg K, Jedrzejczyk D, Vlassis A, Morera-Gómez M, Pereira-Schoning S, Puš U, Oliver-Almirall A, Lyholm Jensen T, Baumgartner R, Tate Weinert B, Gill RT, Warnecke T
Source: ACS Nano
Publication Date: (2023)
Issue: 12(2): 375-389
Research Area:
Cancer Research/Cell Biology
Immunotherapy / Hematology
Molecular Biology
Cells used in publication:
Species: human
Tissue Origin: blood
T cell, human stim.
Species: human
Tissue Origin: blood
4D-Nucleofector® 96-well Systems

 Cell Line Transfection.

A Lonza 4D-Nucleofector with Shuttle unit (V4SC-2960 Nucleocuvette Strips) was used for transfection, following the manufacturer’s instructions. For transfection, cells were harvested by centrifugation (200 × g, RT, 5 min) and re-suspended at 10 × 10^6 cells mL-1 (2 × 10^5 cells 20 µL-1 ) in the SF Cell Line Nucleofector X Kit buffer (Lonza), unless stated otherwise. The cell suspension was mixed with the RNPs, immediately transferred to the nucleocuvette, and transfected using the CA-137 Nucleofector program, except where indicated otherwise. After transfection, the cells were immediately re-suspended in the pre-warmed cultivation medium and plated onto 96-well, flat-bottom, non-treated plates (Falcon) and cultured at 37 °C in 5% CO2 incubators and maintained at a density of 0.5-1.0 × 10^6 cells mL-1. 

Primary T-Cell Transfection.

Forty-eight hours after isolation, the cells were harvested by centrifugation (300 × g, RT, 5 min) and re-suspended at 50 × 10^6 cells mL-1 (1 × 10^6 cells 20 µL-1 ) in the supplemented P3 Primary Cell Nucleofector Kit buffer (Lonza). The cells were mixed with HDRT, and the suspension was transferred to the RNPs immediately before transfection (Nucleofection program EH-115). After transfection, 80 µL of pre-warmed cultivation medium without IL-2 was added to the electroporation cuvettes.


CRISPR-Cas12a nucleases have expanded the toolbox for targeted genome engineering in a broad range of organisms. Here, using a high-throughput engineering approach, we explored the potential of a novel CRISPR-MAD7 system for genome editing in human cells. We evaluated several thousand optimization conditions and demonstrated accurate genome reprogramming with modified MAD7. We identified crRNAs that allow for =95% non-homologous end joining (NHEJ) and 66% frameshift mutations in various genes and observed the high-cleavage fidelity of MAD7 resulting in undetectable off-target activity. We explored the dsDNA delivery efficiency of CRISPR-MAD7, and by using our optimized transfection protocol, we obtained =85% chimeric antigen receptor (CAR) insertions in primary T cells, thus exceeding the baseline integration efficiencies of therapeutically relevant transgenes using currently available virus-free technologies. Finally, we evaluated multiplex editing efficiency with CRISPR-MAD7 and demonstrated simultaneous =35% CAR transgene insertions and =80% gene disruption efficiencies. Both the platform and our transfection procedure are easily adaptable for further preclinical studies and could potentially be used for clinical manufacturing of CAR T cells.