Pooled Knockin Targeting for Genome Engineering of Cellular Immunotherapies

Roth TL, Li PJ, Blaeschke F, Nies JF, Apathy R, Mowery C, Yu R, Nguyen MLT, Lee Y, Truong A, Hiatt J, Wu D, Nguyen DN, Goodman D, Bluestone JA, Ye CJ, Roybal K, Shifrut E, Marson A
Source: Cell Res
Publication Date: (2020)
Issue: 181(3): 728-744.e21
Research Area:
Cancer Research/Cell Biology
Immunotherapy / Hematology
Gene Expression
Cells used in publication:
T cell, human stim.
Species: human
Tissue Origin: blood
Culture Media:
4D-Nucleofector® 96-well Systems

XVivo15 medium (Lonza) supplemented with 5% fetal bovine serum, 50 mM 2-mercaptoethanol, and 10m MN-acetyl L-cystine was used to culture primary human T cells. In preparation for electroporation, T cells were stimulated for 2 days at a starting density of approximately 1 million cells per mL of media with anti-human CD3/CD28 Dynabeads (ThermoFisher), at a bead to cell ratio of 1:1, and cultured in XVivo15 media containing IL-2 (500 U/ml; UCSF Pharmacy), IL-7 (5 ng/ml; ThermoFisher), and IL-15 (5 ng/ml; Life Tech). Following electroporation, T cells were cultured in XVivo15 media containing IL-2 (500 U/ml) and maintained at approximately 1 million cells per mL of media. Every 2-3 days, electroporated T cells were topped up, with or without splitting, with additional media along with additional fresh IL-2 (final concentration of 500 U/ml). When necessary, T cells were transferred to larger culture vessels.

Primary T cell electroporation—For all electroporation experiments, primary T cells were prepared and cultured as described above. After stimulation for 48–56 hours, T cells were collected from their culture vessels and the anti-human CD3/anti-CD28 Dynabeads were magnetically separated from the T cells. Immediately before electroporation, debeaded cells were centrifuged for 10 min at 90g, aspirated, and resuspended in the Lonza electroporation buffer P3. Each experimental condition received a range of 750,000 – 1 million activated T cells resuspended in 20 µL of P3 buffer, and all electroporation experiments were carried out in 96 well format. All electroporations were done using a Lonza 4D 96-well electroporation system with pulse code EH115. Unless otherwise indicated, 3.5 µl RNPs (comprising 50 pmol of total RNP) were electroporated, along with 1–3 µl HDR Template at 1 µg/µl (1–3 µg HDR template total). Immediately after all electroporations, 80 µl of pre-warmed media (without cytokines) was added to each well, and cells were allowed to rest for 15 min at 37 °C in a cell culture incubator while remaining in the electroporation cuvettes. After 15 min, cells were moved to final culture vessels and media supplemented with 500 U/ml IL-2 was added.


Adoptive transfer of genetically modified immune cells holds great promise for cancer immunotherapy. CRISPR knockin targeting can improve cell therapies, but more high-throughput methods are needed to test which knockin gene constructs most potently enhance primary cell functions in vivo. We developed a widely adaptable technology to barcode and track targeted integrations of large non-viral DNA templates and applied it to perform pooled knockin screens in primary human T cells. Pooled knockin of dozens of unique barcoded templates into the T cell receptor (TCR)-locus revealed gene constructs that enhanced fitness in vitro and in vivo. We further developed pooled knockin sequencing (PoKI-seq), combining single-cell transcriptome analysis and pooled knockin screening to measure cell abundance and cell state ex vivo and in vivo. This platform nominated a novel transforming growth factor ß (TGF-ß) R2-41BB chimeric receptor that improved solid tumor clearance. Pooled knockin screening enables parallelized re-writing of endogenous genetic sequences to accelerate discovery of knockin programs for cell therapies.