The successes of genetically engineered chimeric antigen receptor (CAR) T cells in B cell
leukemia and lymphomas have revolutionized the field of immunotherapy. However, durable
remission is only achieved in a subset of patients, and poor treatment efficacy has been
reported for solid tumors. These limitations often result from intrinsic dysfunctions of T cells,
such as poor proliferation, compromised effector function, or T cell exhaustion.
CRISPR-based functional genetic screens assess the impact of genetic modifications at the
genome-wide level and could greatly accelerate the design of improved T cell-based
therapies. We developed a new technology, CRISPR applied by mRNA electroporation and
lentivirus, to enable efficient genome editing and high-throughput screening in CAR T cells.
The simultaneous delivery of the CAR and a CRISPR guide RNA (gDNA) enabled single-
gene perturbation as well as genetic screens with libraries of target genes in CAR T cells
directed against various antigens. Our mRNA-based protocol for the delivery of CRISPR
modifiers extends the available tool kit for genome engineering in primary human T cells
from loss-of-function mutations to precise base editing and CRISPR activation.
We performed genome-scale fitness-based CRISPR screens in human T and CAR T cells. We
uncovered gene regulators specific to TCR-driven and CAR-driven proliferation. Moreover,
our screens robustly identified canonical immunotherapy targets such as CTLA4, along with
genes that have not been characterized in T cells. We have thus demonstrated the high
scalability and efficiency of our technology and created a comprehensive map of genetic
perturbations tuning CAR T cell performance.
Our platform is compatible with CROP-seq technology for single-cell CRISPR screens and
with pooled in vivo screening of CAR T cells in a mouse leukemia model. Thus, the
technology enables unbiased immunotherapy target discovery, and can be applied for high-
throughput characterization of functional regulators in primary human cells.
No relevant conflicts of interest to declare.
