Journal: Nature

Article Title: Systematic discovery of CRISPR-boosted CAR T cell immunotherapies

doi: 10.1038/s41586-025-09507-9

Figure Lengend Snippet: a , Overview of the CELLFIE platform for highly scalable CAR T cell engineering and CRISPR screening. Human primary T cells are isolated, activated and expanded, transduced with a lentivirus carrying sequences for the CAR and gRNA library and electroporated with synthetic mRNA delivering the CRISPR editor. The CRISPR-edited CAR T cells are functionally screened in vitro and in vivo using multiple readouts. hU6, human U6 promoter; LTR, long terminal repeat; puro, puromycin. b , Experimental timeline for genome-wide fitness screens. Human primary T cells are isolated from whole blood, activated, pre-expanded, activated again and transduced with the CROP-seq-CAR lentivirus for co-delivery of sequences for the CAR and the genome-wide gRNA library. Two days later, cells are electroporated with synthetic mRNA for Cas9 and blasticidin resistance (blasticidin-S deaminase, BSD), followed by antibiotic selection for successful lentiviral transduction (puromycin) and successful mRNA electroporation (blasticidin). CRISPR-edited CAR T cells are expanded under repeated TCR stimulation with anti-CD3/CD28 beads or repeated CAR stimulation with CD19 + K562 cells. Genomic DNA is collected, and gRNA representation is analysed by sequencing on days 0, 7, 14 and 21. c , Gene-level log 2 [fold change (FC)] between day 14 and day 0 for the fitness screens (four donors in two independent screens), mapped onto a schematic of the TCR and CAR signalling pathways. For each protein shape, the colour in the top half corresponds to the TCR stimulation screens and the colour in the bottom half corresponds to the CAR stimulation screens. d , Effect sizes for fitness screens with TCR stimulation ( x axis) and CAR stimulation ( y axis). MAGeCK MLE β values comparing day 14 and day 0 are normalized to values for essential genes to account for the different proliferation rates upon TCR or CAR stimulation. Colours denote screening hits with increased fitness (green), known negative T cell regulators (magenta), known essential genes (purple) and neutral olfactory receptors (blue).

Article Snippet: For RNP electroporation, synthetic gRNA (TrueGuide Synthetic gRNA, Synthego) and Alt-R S.p.

Techniques: CRISPR, Isolation, Transduction, In Vitro, In Vivo, Genome Wide, Selection, Electroporation, Sequencing

Journal: Nature

Article Title: Systematic discovery of CRISPR-boosted CAR T cell immunotherapies

doi: 10.1038/s41586-025-09507-9

Figure Lengend Snippet: a , Titration of synthetic mRNA concentration for efficient CRISPR knockout of CD44 in human primary CD4 + and CD8 + T cells (2 donors), with pan-CD3 + T cells as starting material. Comparable editing efficiencies were observed for custom-made and commercial Cas9 mRNA. b , Consistent editing efficiencies in CD4 + T cells for custom-made Cas9 mRNA from multiple production rounds. c , Cell proliferation of CD4 + and CD8 + CAR T cells made from pan-CD3 + T cells (green), from isolated CD4 + T cells (blue), or from isolated CD8 + T cells (purple) as starting material. d , Declining representation of CD4 + CAR T cells in co-culture with CD8 + CAR T cells when using pan-CD3 + T cells as starting material. e , Puromycin titration to determine an optimal concentration (0.5 µg ml −1 , grey dotted line) to select for T cells that have been successfully transduced with the CROP-seq-CAR lentivirus. f , Blasticidin titration to determine an optimal concentration (50 µg ml −1 , grey dotted line) to select for T cells that were successfully electroporated with blasticidin resistance mRNA co-delivered with the CRISPR editor mRNA. g , CD44 knockout efficiency using the optimized electroporation programs for small-scale experiments (up to 1.5 million cells per cuvette, Amaxa electroporator) and large screens (up to 100 million cells per cuvette, MaxCyte electroporator). Results are shown for CD4 + and CD8 + T cells from pan-CD3 + T cells (1 donor). h , T cell expansion after electroporation of synthetic Cas9 mRNA and blasticidin resistance mRNA (1 donor). i , Effect of T cell stimulation reagents on lentiviral transduction rates (4 donors). j , Effect of common transduction supplements on lentiviral transduction rates (4 donors). k , Effect of common transduction supplements on T cell viability (4 donors). l , Quantification of lentivirus titers using RT-qPCR of lentiviral RNA (mean ± s.e.m. for 3 technical replicates). m , Percent transduced CD4 + and CD8 + T cells for a titration of lentivirus amounts. The chosen amounts (CD4 + : 247 copies per cell; CD8 + : 432 copies per cell) are indicated by dotted lines (2 donors). n , Detection of gRNAs (y-axis: gRNA read counts) in clonally expanded human primary T cells transduced with CROP-seq-CAR lentivirus carrying the genome-wide Brunello gRNA library. Representative examples of T cell clones with 1, 2, or 3 gRNA integrations are shown, and a total of 62 clonally expanded T cell clones were profiled. o , Barplot showing the frequency of 1, 2, or 3 independent lentiviral integrations into the same cell across 62 clonally expanded T cell clones. The average number of gRNA integrations per cell was 1.5. p , CAR expression in human primary CAR T cells prepared with the CROP-seq-CAR lentivirus, using PE-labelled recombinant CD19 antigen for labeling. q , Specific killing of CD19 + cancer cells by CAR T cells prepared with the CROP-seq-CAR (anti-CD19) lentivirus. For all boxplots (panels i-l ), the center line is the median, the box limits are the upper and lower quartiles, and the whiskers extend to 1.5 times the interquartile range.

Article Snippet: For RNP electroporation, synthetic gRNA (TrueGuide Synthetic gRNA, Synthego) and Alt-R S.p.

Techniques: Titration, Concentration Assay, CRISPR, Knock-Out, Isolation, Co-Culture Assay, Transduction, Electroporation, Cell Stimulation, Quantitative RT-PCR, Genome Wide, Clone Assay, Expressing, Recombinant, Labeling

Journal: Nature

Article Title: Systematic discovery of CRISPR-boosted CAR T cell immunotherapies

doi: 10.1038/s41586-025-09507-9

Figure Lengend Snippet: a , Proof-of-concept fitness screen with a focused gRNA library of 100 gRNAs. gRNA-level log fold changes are shown for positive control gRNAs (targeting the puromycin resistance gene PAC) and control gRNAs (targeting a safe harbor locus) at days 7, 14, or 21 relative to day 0. Cells were electroporated with custom-made (top) or commercial (bottom) Cas9 mRNA. b , gRNA representation after cloning the genome-wide Brunello library into the CROP-seq-CAR vector, plotted as a cumulative distribution function based on amplicon sequencing of the plasmid pool, with highlighted fold difference between the 10 th and 90 th percentiles as a measure of gRNA library balance. c , Detailed experimental timeline for the genome-wide fitness screens (4 donors, 2 independent experiments). Key steps in the CELLFIE workflow are highlighted. Samples for gRNA sequencing were collected at day 0 (before Cas9 electroporation), and at days 7, 14, and 21 after electroporation. d , Expansion of human primary CAR T cells upon repeated CAR or TCR stimulation (2 donors). e , CD19 accumulation on the surface of CAR T cells during co-culture with target cells as the result of trogocytosis. f , Flow cytometry profiling of the T cell exhaustion markers PD1, LAG3, TIM3, and TIGIT during co-culture of CAR T cells with K562-CD19 target cells. g , T cell subset profiling by flow cytometry at the isolation and readout time points of the genome-wide screens. h , Essential genes in human primary CAR T cells under CAR and TCR stimulation (4 donors, details in Supplementary Table ). The scatterplot shows gene-level log fold changes comparing day 14 and day 0 of the screen (x-axis) plotted against FDR-adjusted p-values (y-axis). Genes that passed stringent significance thresholds (FDR < 0.01 and log FC < − 1.5 based on MAGeCK RRA) are highlighted. i , Gene set enrichment analysis for essential genes in human primary CAR T cells under CAR and TCR stimulation. Clustering of the top-100 most enriched Gene Ontology (GO) terms from the Biological Process category (left) is shown together with one cluster related to T cell functions visualized as a tree plot (right).

Article Snippet: For RNP electroporation, synthetic gRNA (TrueGuide Synthetic gRNA, Synthego) and Alt-R S.p.

Techniques: Positive Control, Control, Cloning, Genome Wide, Plasmid Preparation, Amplification, Sequencing, Electroporation, Co-Culture Assay, Flow Cytometry, Isolation

Journal: Nature

Article Title: Systematic discovery of CRISPR-boosted CAR T cell immunotherapies

doi: 10.1038/s41586-025-09507-9

Figure Lengend Snippet: a , Experimental timeline of the in vivo validation experiments with CRISPR-boosted CAR T cells, which were genetically engineered either by lentiviral co-delivery of the CAR and a pool of 8 gRNAs followed by mRNA delivery of Cas9 (as in the in vitro screens, top) or by electroporation of a pre-assembled RNP complex of Cas9 protein and one top-performing gRNA (as is common practice in CRISPR-edited cell therapy, bottom). b , CAR T cell titration in a xenograft mouse model of human leukemia. Immunodeficient NSG mice were injected with 0.5 million NALM6 cells engineered to express firefly luciferase. On day 5, mice were treated with different doses of CAR T cells. Leukemic cell load was monitored using live bioluminescence imaging. When left untreated, mice succumb to the leukemia around day 21. To make the model most informative, we selected a low (and deliberately non-curative) dose of CAR T cells that leads to initial leukemic control followed by a quick relapse. c , Survival analysis for the mice shown in panel b . d , Percentage of UMI reads perfectly matching the reference sequence, comparing the established method (gRNA amplification from genomic DNA) with in vivo CROP-seq (gRNA amplification from mRNA), both tested with single PCR and nested PCR amplification. e , Optimal number of UMI-based internal replicates for data analysis based on screening controls. Given that each UMI base can be A, C, G, or T, using UMI bases 1 to 5 results in 4, 16, 64, 256, and 1024 random internal replicates. Standard analysis is labeled as 0 internal replicates (left). Negative, neutral, and positive controls are color-coded. f , Dropout of neutral control gRNAs targeting a safe harbor locus when the read number in the internal replicates gets too small for large numbers of internal replicates. The box plot’s center line indicates the median, the box limits represent the upper and lower quartiles, and the whiskers extend to 1.5 times the interquartile range. g , Recall of negative and positive controls for different numbers of internal replicates. The grey line represents the optimal number of 16 internal replicates chosen for the analysis. h , gRNAs selected for in vivo screens (8 gRNAs per gene) or individual validation (as pools of 8 or single gRNAs). i , Individual effects of the 8 gRNAs per gene in the focused validation screen, with single gRNAs used for individual validation highlighted. The box plot’s center line indicates the median, the box limits are the upper and lower quartiles, and the whiskers extend to 1.5 times the interquartile range. j , Number of distinct T cell clones detected based on unique molecular identifiers (UMIs) in the in vivo screens. k , Estimation of CELLFIE’s scalability to large discovery screens in vivo, extrapolating the number of screenable perturbations from empirical measurements for different screening configurations.

Article Snippet: For RNP electroporation, synthetic gRNA (TrueGuide Synthetic gRNA, Synthego) and Alt-R S.p.

Techniques: In Vivo, Biomarker Discovery, CRISPR, In Vitro, Electroporation, Titration, Injection, Luciferase, Imaging, Control, Sequencing, Amplification, Nested PCR, Labeling, Clone Assay