Genome-wide CRISPR Screens in Primary Human T Cells Reveal Key Regulators of Immune Function.

Authors:
Shifrut E, Carnevale J, Tobin V, Roth TL, Woo JM, Bui CT, Li PJ, Diolaiti ME,  Ashworth A, Marson A. 
In:
Source: Cell
Publication Date: (2018)
Issue: 175(7): 1985-1971
Research Area:
Immunotherapy / Hematology
Gene Expression
Cells used in publication:
T cell, human stim.
Species: human
Tissue Origin: blood
PBMC, human
Species: human
Tissue Origin: blood
Culture Media:
Platform:
96-well Shuttle™ System
4D-Nucleofector™ X-Unit
Experiment

CD8+ T-cells Cells were electroporated at 20e6 cells per cuvette using the pulse code EH115 . The total number of cells for electroporation
was scaled as required. Immediately after electroporation, 1mL of pre-warmed media was added to each cuvette and cuvettes were
placed at 37 degrees for 20 minutes. Cells were then transferred to culture vessels in X-Vivo media containing 50U/mL IL-2 at
1e6 cells /mL
in appropriate tissue culture vessels. Cells were expanded every two days, adding fresh media with IL-2 at 50U/mL
and maintaining the cell density at 1e6 cells /mL.

Assembled ribonucleotide proteins (RNPs) were dispensed into a 96W V-bottom plate at 3mL per well. Cells
were spun down, resuspended in Lonza P3 buffer at 1e6 cells per 20mL, and added to a V-bottom plate with RNPs. The cells/RNP
mixture was transferred to a 96 well electroporation cuvette plate (Lonza, cat #VVPA-1002) for nucleofection using the pulse code
EH115. Immediately after electroporation, 80mL of pre-warmed media was added to each well and incubated at 37C for 20 minutes.

48 hours after transduction, 1e6 cells were resuspended in P3 buffer and 3 mL of Cas9 (Stock 40 mM) was added. Cells were transferred
to a 96 well electroporation cuvette plate (Lonza, cat #VVPA-1002) for nucleofection using the pulse code EH115. 24 hours post
nucleofection, cells were treated with 2.5ug/mL Puromycin for three days, and subsequently sorted for live cells using Ghost Dye 710
(Tonbo Biosciences, Cat #13-0871).

Abstract

Human T cells are central effectors of immunity and cancer immunotherapy. CRISPR-based functional studies in T cells could prioritize novel targets for drug development and improve the design of genetically reprogrammed cell-based therapies. However, large-scale CRISPR screens have been challenging in primary human cells. We developed a new method, single guide RNA (sgRNA) lentiviral infection with Cas9 protein electroporation (SLICE), to identify regulators of stimulation responses in primary human T cells. Genome-wide loss-of-function screens identified essential T cell receptor signaling components and genes that negatively tune proliferation following stimulation. Targeted ablation of individual candidate genes characterized hits and identified perturbations that enhanced cancer cell killing. SLICE coupled with single-cell RNA sequencing (RNA-seq) revealed signature stimulation-response gene programs altered by key genetic perturbations. SLICE genome-wide screening was also adaptable to identify mediators of immunosuppression, revealing genes controlling responses to adenosine signaling. The SLICE platform enables unbiased discovery and characterization of functional gene targets in primary cells.