Comment[GEAAccession]	E-GEAD-890
MAGE-TAB Version	1.1
Investigation Title	Genome-wide CRISPR screen in human T cells reveals regulators of FOXP3 [RNA-seq of resting cells]
Experiment Description	Regulatory T cells (Tregs), which specifically express the master transcription factor FoxP3, play a pivotal role in maintaining immunological tolerance and homeostasis and have the potential to revolutionize cell therapies for autoimmune diseases. While stimulation of naive CD4+ T cells in the presence of TGF-beta and IL-2 can induce FoxP3+ Tregs in vitro (iTregs), the resulting cells are often unstable and have thus far hampered translational efforts. A systematic approach towards understanding the regulatory networks that dictate Treg differentiation could lead to more effective iTreg cell-based therapies. Here we performed a genome-wide CRISPR loss-of-function screen to catalog gene regulatory determinants of FOXP3 induction in primary human T cells and characterized their effects at single-cell resolution using Perturb-icCITE-seq. Our findings reveal novel regulators of FOXP3 and point towards new avenues to improve the efficacy of adoptive cell therapy for autoimmune disease.
Experimental Design	genetic modification design
Experimental Factor Name	genetic_modification
Experimental Factor Type	genetic_modification
Person Last Name	Takeshima
Person First Name	Yusuke
Person Affiliation	Osaka University, WPI Immunology Frontier Research Center
Person Roles	submitter
Public Release Date	2025-01-07
PubMed ID	40140585
Protocol Name	P-GEAD-3442	P-GEAD-3443	P-GEAD-3444	P-GEAD-3445	P-GEAD-3446
Protocol Type	sample collection protocol	nucleic acid extraction protocol	nucleic acid library construction protocol	nucleic acid sequencing protocol	normalization data transformation protocol
Protocol Description	Naive CD4 Tconv cells were sorted from PBMC (Day 0). Cells were stimulated with plate-bound anti-human CD3 (10 ug/mL; clone UCHT1) for 72 hours. Cells were collected and resuspended with freshly prepared medium including 500 IU/mL of hIL-2 (Day 3) and expanded for 6 days. 400 uM of CRISPR RNAs (crRNAs) and trans-activating CRISPR RNAs (tracrRNAs) (Integrated DNA Technologies) were mixed at a 1:1 vol/vol ratio and heated at 95C for 5 min. Following an incubation period of 15 minutes at room temperature, 30_ug of Cas9 protein (Takara, #632641) was added to 2.7 uL of 200 uM gRNA complex, and then incubated for over 15 min at room temperature before use. Pooled RNPs with two different gRNAs were also used in this study to maximize knockout efficiency of the target protein. In this case, half the amount of gRNA complex and Cas9 protein were used for preparing each RNP complex and then mixed at a 1:1 ratio. For electroporation, pre-activated CD4-positive T cells were resuspended in P2 primary nucleofection buffer at 2x10E6 cells/20 uL and mixed with RNPs in a 16-well cuvette plate. The cell-containing mixture was pulsed with the EH-100 program and electroporated cells were cultured at 1-2x10E6 cells/mL in complete medium supplemented with 500 IU/mL IL-2 for 4-6 days.	Total RNA was extracted using the miRNeasy micro kit (Qiagen). The quality of the RNA was assessed using an RNA 6000 Pico Kit on the 2100 Bioanalyzer System (Agilent Technologies). All sequenced samples had an RNA integrity number (RIN) of >8.	RNA-sequencing libraries were prepared using the Smart-seq HT kit (Takara; 634455) in combination with the Nextera XT DNA Library Preparation Kit (Illumina; FC-131-1096) following the manufacturer's instructions.	Prepared libraries were sequenced on the NovaSeq platform (Illumina).	Raw fastq files were aligned to the human genome (GRCh38; GenBank assembly GCA_000001405.40) using the STAR1 pipeline (v2.7.10b). HTSeq-count2 (v2.0.2) was used to generate gene counts and genes were filtered to retain those with raw read counts >= 10 in at least 2 samples. The resulting gene expression matrix was subsequently normalized using the trimmed mean of M value (TMM) method.
SDRF File	E-GEAD-890.sdrf.txt
Comment[AEExperimentType]	RNA-seq of coding RNA
Comment[BioProject]	PRJDB16517
Comment[Last Update Date]	2025-08-13