Large-scale CRISPR screening in primary human 3D gastric organoids enables comprehensive dissection of gene-drug interactions

Depletion of tumor suppressor LRIG1 was identified as the top hit contributing to increased cell proliferation.

“Notably, the depletion of tumor suppressor LRIG1, a negative regulator of ERBB receptor tyrosine kinases40–42, was identified as the top hit contributing to increased cell proliferation (Fig. 1D).”

• cites [40] Powell, A. E. et al. The pan-ErbB negative regulator Lrig1 is an intestinal stem cell marker that functions as a tumor suppressor. Cell 149, 146–158 (2012). sourceSupported
• cites [41] Wang, Y. et al. Loss of Lrig1 leads to expansion of Brunner glands followed by duodenal adenomas with gastric metaplasia. Am. J. Pathol. 185, 1123–1134 (2015). sourceSupported
• cites [42] Yu, S. et al. Expression of LRIG1, a negative regulator of EGFR, is dynamically altered during different stages of gastric carcinogenesis. Am. J. Pathol. 188, 2912–2923 (2018). sourceSupported

Several mRNA 3′ end processing factors and RNA-binding proteins, such as CPSF2, ZFP36L2, and SRSF7, were identified among cisplatin sensitization hits.

“In addition, in agreement with the importance of pre-mRNA processing factors in the DNA damage response46, several mRNA 3′ end processing factors and RNA-binding proteins were identified, such as CPSF2, ZFP36L2, and SRSF7.”

• cites [46] Montecucco, A. & Biamonti, G. Pre-mRNA processing factors meet the DNA damage response. Front. Genet. 4, 102 (2013). sourceSupported

ELOF1 and LEO1 were identified among cisplatin sensitization hits.

“Notably, ELOF1, a recently identified transcription elongation factor that plays an important role in transcription-coupled nucleotide-excision repair47, and LEO1, a subunit of the polymerase-associated factor 1 (Paf1) complex RNA polymerase that is required for RNA polymerase II removal in response to DNA damage, were identified48,49.”

• cites [47] van der Weegen, Y. et al. ELOF1 is a transcription-coupled DNA repair factor that directs RNA polymerase II ubiquitylation. Nat. Cell Biol. 23, 595–607 (2021). sourcePartly supported

  In Results, ‘A CRISPR screen identifies ELOF1 as a putative TCR gene’ (page not provided), ELOF1 was a hit in an Illudin S screen, and ELOF1-knockout cells were sensitive to cisplatin in a separate drug-sensitivity assay. The cited paper does not establish that ELOF1 was a cisplatin-screen hit or identify LEO1 as one. (LLM, conf=0.85)

  Experimental — accuracy not yet measured
• cites [48] Tiwari, V., Kulikowicz, T., Wilson, D. M. 3rd & Bohr, V. A. LEO1 is a partner for Cockayne syndrome protein B (CSB) in response to transcription-blocking DNA damage. Nucleic Acids Res. 49, 6331–6346 (2021). sourcePartly supported

  In “LEO1 deficiency increases sensitivity to certain genotoxins” (page not available), the paper reports that LEO1-deficient cells were significantly more sensitive to cisplatin. It does not identify ELOF1 or report that either gene was a hit in a cisplatin-sensitization screen; the citing sentence appears to present that identification as the citing authors’ own finding. (LLM, conf=0.87)

  Experimental — accuracy not yet measured
• cites [49] Chen, F., Liu, B., Zhou, H. & Long, J. The Paf1 complex is required for RNA polymerase II removal in response to DNA damage. Proc. Natl. Acad. Sci. USA 119, e2207332119 (2022). sourceCould not verify

  The Results sections on Paf1/Leo1 describe Leo1’s role in RNA polymerase II removal after DNA damage, but do not report a cisplatin-sensitization screen or identify ELOF1 and LEO1 as hits. Searches for cisplatin sensitivity, sensitization hits, and ELOF1 found no passage addressing that claim. The cited paper does support the separate description of LEO1’s role in the citing sentence. (LLM, conf=0.73)

  Experimental — accuracy not yet measured

The TP53/APC double knockout (DKO) organoid line was established by sequentially disrupting TP53 and APC from non-neoplastic human gastric organoids.

“The TP53/APC double knockout (DKO) organoid line was established in our previous studies by sequentially disrupting TP53 and APC37, two common oncogenic loci in gastric adenocarcinoma38,39, from non-neoplastic human gastric organoids (Fig. S1A).”

• cites [37] Karlsson, K. et al. Deterministic evolution and stringent selection during preneoplasia. Nature 618, 383–393 (2023). sourcePartly supported

  In “TP53–/– induces CNAs in defined orders” (page not provided), the paper says it established organoids from non-malignant human gastric tissue and engineered TP53 and APC inactivation in two cultures. It describes the APC hit as “concurrently engineered,” however, so it does not support the claimed sequential disruption. (LLM, conf=0.87)

  Experimental — accuracy not yet measured
• cites [38] Cancer Genome Atlas Research Network. Comprehensive molecular characterization of gastric adenocarcinoma. Nature 513, 202–209 (2014). sourceSupported
• cites [39] Cristescu, R. et al. Molecular analysis of gastric cancer identifies subtypes associated with distinct clinical outcomes. Nat. Med. 21, 449–456 (2015). sourceNot checked — no full text of the source

Cas9 integration enhances CRISPR knockout efficiency in gastric organoids.

“Building on our previous study that Cas9 integration enhances CRISPR knockout efficiency in gastric organoids9, we first generated stable Cas9-expressing TP53/APC DKO organoids”

• cites [9] Lo, Y. H. et al. A CRISPR/Cas9-engineered ARID1A-deficient human gastric cancer organoid model reveals essential and nonessential modes of oncogenic transformation. Cancer Discov. 11, 1562–1581 (2021). sourceSupported

Many genes identified from iCRISPRi pooled screens in 3D human gastric organoids were not determined as cisplatin sensitization partners in previous genome-scale CRISPR KO screens in immortalized 2D human cell lines.

“In particular, many genes identified from our iCRISPRi pooled screens in 3D human gastric organoids were not determined as cisplatin sensitization partners in previous genome-scale CRISPR KO screens in immortalized 2D human cell lines51, suggesting potential 3D specificity (Fig. S6A).”

• cites [51] Olivieri, M. et al. A genetic map of the response to DNA damage in human cells. Cell 182, 481–496.e421 (2020). sourceSupported

High sgRNA and transcript capture rates indicated comparable quality in organoids to previous datasets in various cell lines.

“Moreover, high sgRNA and transcript capture rates indicated a comparable quality in organoids to previous datasets in various cell lines32,50 (Fig. S5D, E).”

• cites [32] Adamson, B. et al. A multiplexed single-cell CRISPR screening platform enables systematic dissection of the unfolded protein response. Cell 167, 1867–1882.e1821 (2016). sourceSupported
• cites [50] Replogle, J. M. et al. Combinatorial single-cell CRISPR screens by direct guide RNA capture and targeted sequencing. Nat. Biotechnol. 38, 954–961 (2020). sourceSupported

Recent studies have shown that phenotypes associated with CRISPR-mediated knockout of putative oncogenic loci are more pronounced when cancer cells are grown as 3D spheroids than 2D monolayers.

“Recent studies have shown that phenotypes associated with CRISPR-mediated knockout of putative oncogenic loci are more pronounced when cancer cells are grown as 3D spheroids than 2D monolayers, suggesting that 3D culture systems may be particularly useful for uncovering cancer vulnerabilities55.”

• cites [55] Han, K. et al. CRISPR screens in cancer spheroids identify 3D growth-specific vulnerabilities. Nature 580, 136–141 (2020). sourceSupported

CRISPRa-based screens are inherently less reproducible than CRISPRi or knockout screens.

“In addition, CRISPRa-based screens are inherently less reproducible than CRISPRi or knockout screens43.”

• cites [43] Sanson, K. R. et al. Optimized libraries for CRISPR-Cas9 genetic screens with multiple modalities. Nat. Commun. 9, 5416 (2018). sourcePartly supported

  The Results section (‘Optimized genome-wide CRISPRa library’; page unavailable) reports low replicate correlations in specific CRISPRa resistance screens, but says low correlation is generally expected in positive-selection screens. It does not establish that CRISPRa is inherently less reproducible than CRISPRi or knockout screens. (LLM, conf=0.85)

  Experimental — accuracy not yet measured

Our oncogene-engineered human tumor organoids represent a pre-cancerous stage.

“Finally, our oncogene-engineered human tumor organoids represent a pre-cancerous stage9,37, meaning the gene-specific dependencies and biological processes identified may be distinct to early cancer development as opposed to established tumors.”

• cites [9] Lo, Y. H. et al. A CRISPR/Cas9-engineered ARID1A-deficient human gastric cancer organoid model reveals essential and nonessential modes of oncogenic transformation. Cancer Discov. 11, 1562–1581 (2021). sourceSupported
• cites [37] Karlsson, K. et al. Deterministic evolution and stringent selection during preneoplasia. Nature 618, 383–393 (2023). sourcePartly supported

  The Main section describes CRISPR-engineered human gastric organoids used to model preneoplasia, and the Discussion says the TP53-deficient organoids recapitulate occult preneoplasia. That supports the use of engineered human organoids as a pre-cancer model, but the paper does not establish that the citing authors’ own oncogene-engineered tumor organoids are at that stage. (LLM, conf=0.82)

  Experimental — accuracy not yet measured

There is an unexpected link between fucosylation and cisplatin sensitivity.

“uncover an unexpected link between fucosylation and cisplatin sensitivity”

Combining CRISPR perturbations with single-cell transcriptomics resolves how genetic alterations interact with cisplatin at the level of individual cells.

“By combining CRISPR perturbations with single-cell transcriptomics, we resolve how genetic alterations interact with cisplatin at the level of individual cells”

The screens uncover genes that modulate cisplatin response.

“Our screens uncover genes that modulate cisplatin response.”

Large-scale CRISPR-based genetic screens, including knockout, CRISPRi, CRISPRa, and single-cell approaches, can be applied in primary human 3D gastric organoids to systematically identify genes that affect sensitivity to cisplatin.

“Here we show that large-scale CRISPR-based genetic screens, including knockout, interference (CRISPRi), activation (CRISPRa), and single-cell approaches, can be applied in primary human 3D gastric organoids to systematically identify genes that affect sensitivity to cisplatin.”

TAF6L is a regulator of cell recovery from cisplatin-induced cytotoxicity.

“We identify TAF6L as a regulator of cell recovery from cisplatin-induced cytotoxicity.”

iCRISPRi-sgCXCR4 organoids exhibited a decreased CXCR4-positive population (3.3%) compared to parental iCRISPRi organoids (13.1%) five days post-induction.

“Five days post-induction, iCRISPRi-sgCXCR4 organoids exhibited a decreased CXCR4-positive population (3.3%) compared to the parental iCRISPRi organoids (13.1%) (Fig. 2D).”

A systematic approach enables a full suite of high-representation CRISPR-based genetic screens (CRISPR cutting, CRISPRi, CRISPRa, and single-cell CRISPR screens) to study gene-drug interactions in organoids using oncogene-engineered human gastric tumor models.

“Here, we report a systematic approach that enables a full suite of high-representation CRISPR-based genetic screens, including CRISPR cutting, CRISPRi, CRISPRa, and single-cell CRISPR screens, to study gene-drug interactions in organoids, using oncogene-engineered human gastric tumor models.”

• AssumptionRests on an assumption: The feasibility of using CRISPR screens in 3D human organoids to investigate gene-drug interactions remains to be fully established.

The data reveal DNA repair pathway-specific transcriptomic convergence in cisplatin-treated organoids, manifested by distinct high-dimensional gene expression profiles and growth phenotypes.

“Coupled with single-cell CRISPR screens, our data reveal DNA repair pathway-specific transcriptomic convergence in cisplatin-treated organoids, manifested by distinct high-dimensional gene expression profiles and growth phenotypes.”

Only a few genes showed increased sgRNA abundance, indicating a growth advantage phenotype upon knockout.

“In contrast, only a few genes showed increased sgRNA abundance, indicating a growth advantage phenotype upon knockout (Supplementary Data 2).”

The CXCR4-positive population increased in iCRISPRa-sgCXCR4 organoids (57.6%).

“In contrast, the CXCR4-positive population increased in iCRISPRa-sgCXCR4 organoids (57.6%) (Fig. 2D).”

The study identifies previously unappreciated genes that contribute to cell growth and sensitivity to cisplatin in gastric cancers.

“Specifically, we identify previously unappreciated genes that contribute to cell growth and sensitivity to the chemotherapy drug cisplatin in gastric cancers.”

TAF6L is a key gene involved in cell proliferation during the recovery phase following cisplatin-induced DNA damage.

“We identified TAF6L as a key gene involved in cell proliferation during the recovery phase following cisplatin-induced DNA damage.”

The 68 drop-out genes were enriched in pathways related to essential biological processes such as transcription, RNA processing, and nucleic acid metabolic processes.

“These genes were enriched in pathways related to essential biological processes such as transcription, RNA processing, and nucleic acid metabolic processes (Fig. 1E).”

The iCRISPRa screen identified 9 cisplatin sensitization genes whose overexpression conferred drug sensitivity.

“After this normalization, we identified 9 cisplatin sensitization genes whose overexpression in the iCRISPRa screen conferred drug sensitivity (Figs. 3C and S3B; Supplementary Data 6).”

Inhibition of DNA repair genes ERCC6, ERCC4, and GTF2H5 conferred cisplatin sensitivity.

“As expected, inhibition of DNA repair genes, such as ERCC6, ERCC4, and GTF2H5, conferred cisplatin sensitivity (Fig. 3E).”

There is an unexpected functional connection between protein fucosylation and cisplatin sensitivity.

“We uncovered an unexpected functional connection between protein fucosylation and cisplatin sensitivity.”

Individual sgRNAs targeting the SOX2 promoter were sufficient to inhibit or enhance SOX2 expression in iCRISPRi and iCRISPRa organoids, respectively.

“Moreover, individual sgRNAs targeting the promoter of the transcription factor SOX2 were sufficient to inhibit (iCRISPRi-sgSOX2) or enhance (iCRISPRa-sgSOX2) SOX2 expression (Fig. 2E).”

14 genes among the cisplatin sensitization hits were key components of DNA repair pathways, including nucleotide-excision repair (ERCC6, ERCC4, XPA, ELOF1, GTF2H5, ERCC8), homologous recombination repair (BRCA1, HELQ, MCM9), and Fanconi anemia (SETD1A, BRCA2, FAN1, BOD1L1) pathways.

“14 genes were key components of DNA repair pathways, including nucleotide-excision repair (NER genes, ERCC6, ERCC4, XPA, ELOF1, GTF2H5, and ERCC8), homologous recombination repair (HRR genes, BRCA1, HELQ, and MCM9), and Fanconi anemia (FA genes, SETD1A, BRCA2, FAN1, and BOD1L1) pathways.”

The cisplatin-naïve iCRISPRi screen yielded 394 hits (384 growth defects and 10 growth advantages) that exhibited a cell growth phenotype.

“The cisplatin-naïve iCRISPRi screen yielded 394 hits (384 growth defects and 10 growth advantages) that exhibited a cell growth phenotype (Fig. 3B; Supplementary Data. 5).”

Control sgRNAs were clustered around zero (no phenotype) in the gene-level phenotype score.

“The gene-level phenotype score (see “Methods”) indicated that control sgRNAs were clustered around zero (no phenotype) (Fig. 1D).”

ELOF1, LEO1, and ZNF677 were reconfirmed as cisplatin sensitization hits.

“Moreover, ELOF1, LEO1, and a previously underexplored gene, ZNF677, from the primary screen were successfully reconfirmed as cisplatin sensitization hits (Fig. 3F).”

Depletion of CD151, KIAA1524, TEX10, and RPRD1B reproduced the growth defect phenotype compared to control organoids.

“Compared to control organoids harboring a negative control sgRNA, the depletion of all four selected genes reproduced the growth defect phenotype (Figs. 1F and S1D).”

Among the growth defect hits, gene sets controlling essential biological processes such as RNA catabolism, telomere maintenance, DNA biosynthesis, and cell cycle were enriched.

“Among the growth defect hits, gene sets controlling essential biological processes, such as RNA catabolism, telomere maintenance, DNA biosynthesis, and cell cycle, were enriched (Fig. S2E).”

A significant reduction of the same gene sets was often observed in individual CRISPRa cells exhibiting growth defects, such as those with ZBTB7A and HHEX sgRNAs.

“As expected, a significant reduction of the same gene sets was often observed in individual CRISPRa cells exhibiting growth defects, such as those with ZBTB7A and HHEX sgRNAs (Fig. 4F).”

TAF6L knockdown reduced the EdU+ cell population to an average of 8.0% compared to control organoids (18.1% EdU+ on average).

“However, TAF6L knockdown reduced the EdU+ cell population to an average of 8.0% compared to control organoids (18.1% EdU+ on average), indicating that TAF6L depletion inhibited cell proliferation.”

A strong defective signature of purine biosynthetic pathways was identified in iCRISPRa-sgHHEX cells, suggesting a potential metabolic component underlying HHEX overexpression-induced S phase arrest.

“Notably, a strong defective signature of purine biosynthetic pathways was identified in iCRISPRa-sgHHEX cells, suggesting a potential metabolic component underlying HHEX overexpression-induced S phase arrest (Fig. 4F).”

Under TAF6L knockdown conditions, only about 1% of cells remained EdU+ following cisplatin treatment.

“Under TAF6L knockdown conditions, only about 1% of cells remained EdU+ following cisplatin treatment.”

TAF6L knockdown increased sensitivity to cisplatin compared to control organoids carrying a non-targeting sgRNA.

“Consistent with the primary iCRISPRi screen, TAF6L knockdown increased sensitivity to cisplatin compared to the control organoids carrying a non-targeting sgRNA (Fig. 6B).”

GMDS expression effectively rescued the suppressed GMDS levels in cisplatin-treated organoids.

“Furthermore, GMDS expression effectively rescued the suppressed GMDS levels in cisplatin-treated organoids (Fig. S7E) and resulted in GMDS OE organoids exhibiting an increased sensitivity to cisplatin treatment (Fig. 5G), accompanied by an increase in nuclear phospho-H2AX (γH2AX) (Fig. 5H).”

GO enrichment analysis of upregulated genes in TAF6L knockdown organoids revealed key biological processes such as triglyceride metabolic process, cholesterol transport, and endoplasmic reticulum lumen.

“GO enrichment analysis of the upregulated genes in TAF6L knockdown organoids revealed several key biological processes, such as triglyceride metabolic process, cholesterol transport, and endoplasmic reticulum lumen (Fig. S9A).”

Ectopic expression of TAF6L-GFP protected the organoids from cisplatin-induced cell death.

“As expected, ectopic expression of TAF6L-GFP protected the organoids from cisplatin-induced cell death (Fig. 6D).”

No significant changes in RAD51, FANCD2, or ubiquitinated FANCD2 (Ub-FANCD2) expression were observed in TAF6L knockdown or TAF6L-GFP organoids.

“However, we did not observe significant changes in RAD51, FANCD2, or ubiquitinated FANCD2 (Ub-FANCD2) expression in TAF6L knockdown or TAG6L-GFP organoids (Fig. S8B, C), suggesting that TAF6L does not directly regulate DNA repair signaling activity.”

GMDS OE organoids did not compromise the baseline viability of parental organoids in the absence of cisplatin.

“Notably, GMDS OE organoids did not compromise the baseline viability of parental organoids in the absence of cisplatin (Fig. S7F), further ruling out nonspecific toxicity associated with elevated GMDS levels.”

The combination of ERCC4/cisplatin, ERCC6/cisplatin, and ERCC8/cisplatin all synergistically and profoundly repressed GMDS upon single-cell CRISPR analysis to a much greater extent than predicted by the additive model.

“Interestingly, the combination of ERCC4/cisplatin, ERCC6/cisplatin, and ERCC8/cisplatin all synergistically and profoundly repressed GMDS upon single-cell CRISPR analysis to a much greater extent than predicted by the additive model (Figs. 5C and S7A; Supplementary Data 9).”

• AssumptionThe authors state this as an assumption: The additive model (sum of single-perturbation effects) is an appropriate null for identifying synergistic gene-drug interactions.

TAF6L did not cluster with NER- or HRR-specific groups in single-cell CRISPR results.

“This was consistent with the single-cell CRISPR results (Fig. 5), as TAF6L did not cluster with NER- or HRR-specific groups, indicating that other mechanisms may be at play.”

Heatmap hierarchical clustering of transcriptional profiles revealed correlations of individual genetic perturbations, uncovering functional gene clusters.

“Finally, the heatmap hierarchical clustering of transcriptional profiles revealed correlations of individual genetic perturbations, uncovering functional gene clusters (Fig. 4C).”

Among significantly reduced ATAC peaks in promoter regions, the peak located at the TAF6L promoter ranked as the 1st peak.

“Of note, among the significantly reduced ATAC peaks in promoter regions, the peak located at the TAF6L promoter ranked as the 1st peak (Fig. S9D), consistent with a strong autoregulatory effect at the TAF6L promoter.”

The results demonstrated the feasibility of conducting large-scale CRISPR screens with high representation (>1000× coverage) in human 3D organoids across three different CRISPR platforms: CRISPR cutting, CRISPRi, and CRISPRa.

“Our results demonstrated the feasibility of conducting large-scale CRISPR screens with high representation (>1000× coverage) in human 3D organoids across three different CRISPR platforms: CRISPR cutting, CRISPRi, and CRISPRa.”

The combination of ERCC4 knockdown and cisplatin treatment strongly inhibited reactivity to fucose-recognizing AAL and UEA-I, revealing a decrease of total cellular fucosylated proteins.

“Consistent with the single-cell CRISPR screening result, the combination of ERCC4 knockdown and cisplatin treatment strongly inhibited reactivity to fucose-recognizing Aleuria aurantia lectin (AAL) (Fig. 5D) and Ulex europaeus agglutinin I (UEA-I) (Fig. S7C), revealing a decrease of total cellular fucosylated proteins.”

• AssumptionRests on an assumption: GDP-fucose is the sole donor for protein fucosylation, a critical glycan modification for numerous membrane and secreted proteins.

UMAP analysis indicated distinct clusters grouped by individual targeting sgRNAs.

“The UMAP analysis indicated distinct clusters, grouped by individual targeting sgRNAs (Fig. 4D).”

GMDS OE organoids showed an increase in nuclear phospho-H2AX (γH2AX).

“Furthermore, GMDS expression effectively rescued the suppressed GMDS levels in cisplatin-treated organoids (Fig. S7E) and resulted in GMDS OE organoids exhibiting an increased sensitivity to cisplatin treatment (Fig. 5G), accompanied by an increase in nuclear phospho-H2AX (γH2AX) (Fig. 5H).”

Cisplatin-treated organoids exhibited highly convergent transcriptional signatures of NER or HRR pathways upon inhibition of cisplatin sensitization genes.

“By contrast, cisplatin-treated organoids exhibited highly convergent transcriptional signatures of NER or HRR pathways upon inhibition of cisplatin sensitization genes.”

Several GO terms related to cell proliferation were identified in TAF6L knockdown or TAF6L-GFP organoids.

“However, in agreement with our conclusion that TAF6L regulates cell proliferation, several GO terms related to cell proliferation were identified (Fig. S9C).”

ZKSCAN2 knockdown causes a growth defect phenotype.

“To identify potential previously uncharacterized genes involved in cisplatin sensitivity, we focused on TAF6L, as it is the highest-ranking unreported cisplatin sensitization hit identified from our CRISPR screens (Fig. 3D), aside from ZKSCAN2, whose knockdown causes a growth defect phenotype (Fig. S4B).”

GSEA revealed enrichment of translation, DNA replication, cell cycle progression, gene expression, and telomere maintenance in MYC-overexpressing single cells.

“In addition, consistent with the growth advantage phenotype of MYC-overexpressing single cells, the gene set enrichment analysis (GSEA) revealed enrichment of translation, DNA replication, cell cycle progression, gene expression, and telomere maintenance (Fig. 4F).”

The cisplatin-naïve organoids in the iCRISPRi single-cell CRISPR screen revealed substantial heterogeneity upon individual genetic knockdowns.

“The cisplatin-naïve organoids in the iCRISPRi single-cell CRISPR screen revealed substantial heterogeneity upon individual genetic knockdowns (Fig. 5A).”

Cells transduced with sgRNAs targeting MYC showed increased mitotic M-phase populations.

“By decomposing the cell cycle states of individual cells based on single-cell transcriptomes, we observed increased mitotic M-phase populations in cells transduced with sgRNAs targeting MYC, consistent with its known oncogenic properties (Fig. 4E).”

No significant GO terms related to the DNA damage response were identified in TAF6L knockdown or TAF6L-GFP organoids.

“We did not identify any significant GO terms related to the DNA damage response, suggesting that TAF6L does not directly regulate the expression of DNA repair genes or other related genes.”

TAF6L-GFP expression did not cause notable changes in chromatin accessibility, with only 7 ATAC peaks increasing and 28 decreasing.

“In contrast, TAF6L-GFP expression did not cause notable changes in chromatin accessibility, with only 7 ATAC peaks increasing and 28 decreasing (Fig. 6G; Supplementary Data 13).”

After 48 h of cisplatin treatment, the EdU+ cell population significantly decreased from an average of 18.1% to 3.6% in control organoid lines.

“After 48 h of cisplatin treatment, the EdU+ cell population significantly decreased from an average of 18.1% to 3.6% in control organoid lines (Figs. 6E and S8D).”

GMDS expression was sufficient to induce total cellular protein fucosylation.

“As expected, GMDS expression was sufficient to induce total cellular protein fucosylation (Fig. 5F).”

• AssumptionRests on an assumption: GDP-fucose is the sole donor for protein fucosylation, a critical glycan modification for numerous membrane and secreted proteins.

Cisplatin-treated transcriptomes of ERCC4, ERCC6, ERCC8, GTF2H5, and ELOF1 iCRISPRi knockdown cells strongly clustered.

“Indeed, cisplatin-treated transcriptomes of the ERCC4, ERCC6, ERCC8, GTF2H5, and ELOF1 iCRISPRi knockdown cells strongly clustered (Fig. 5B), consistent with the common involvement of these genes in NER.”

After doxycycline induction, TAF6L expression was reduced by 85% in the stable TAF6L iCRISPRi knockdown organoid line.

“After doxycycline induction, TAF6L expression was reduced by 85% (Fig. 6A).”

The analysis identified numerous gene-drug interactions in which the sgRNA and cisplatin combination synergistically repressed or activated downstream loci much more in actual scRNA-seq data than predicted by the additive model.

“This analysis identified numerous gene-drug interactions in which the sgRNA and cisplatin combination synergistically repressed or activated downstream loci much more in the actual scRNA-seq data (condition 4) than would be predicted by the additive model (condition 3).”

• AssumptionThe authors state this as an assumption: The additive model (sum of single-perturbation effects) is an appropriate null for identifying synergistic gene-drug interactions.

TAF6L is the highest-ranking unreported cisplatin sensitization hit identified from the CRISPR screens, aside from ZKSCAN2.

“To identify potential previously uncharacterized genes involved in cisplatin sensitivity, we focused on TAF6L, as it is the highest-ranking unreported cisplatin sensitization hit identified from our CRISPR screens (Fig. 3D), aside from ZKSCAN2, whose knockdown causes a growth defect phenotype (Fig. S4B).”

Cisplatin treatment decreased GMDS expression.

“Consistent with the Perturb-seq data (Fig. 5C), cisplatin treatment decreased GMDS expression (Fig. 5E).”

Upon hierarchical clustering, the transcriptomes of NER and HRR locus knockdown cells in cisplatin-naïve organoids were substantially intermixed rather than segregating to NER- or HRR-specific clusters.

“In fact, upon hierarchical clustering, the transcriptomes of NER and HRR locus knockdown cells were substantially intermixed rather than segregating to NER- or HRR-specific clusters (Fig. 5A).”

GMDS was also synergistically repressed by the combination of cisplatin with either ZKSCAN2 or LEO1 sgRNA.

“As GMDS was also synergistically repressed by the combination of cisplatin with either ZKSCAN2 or LEO1 sgRNA, it is conceivable that these loci are unsuspected participants in NER.”

TAF6L knockdown resulted in a dramatic decrease in ATAC-seq peaks compared with control iCRISPRi organoids carrying non-targeting sgRNA.

“Surprisingly, TAF6L knockdown resulted in a dramatic decrease in ATAC-seq peaks compared with control iCRISPRi organoids carrying non-targeting sgRNA.”

Top GO terms of downregulated genes in TAF6L knockdown cells included maintenance of gastrointestinal epithelium and epithelium development and differentiation.

“Moreover, the top GO terms of the downregulated genes in TAF6L knockdown cells included maintenance of gastrointestinal epithelium and epithelium development and differentiation (Fig. S9A).”

TAF6L-GFP expression enabled cells to maintain their ability to proliferate after cisplatin treatment, with an average of 4.8% EdU+ post-treatment.

“In contrast, TAF6L-GFP expression enabled cells to maintain their ability to proliferate after cisplatin treatment, with an average of 4.8% EdU+ post-treatment.”

Re-expression of TAF6L-GFP was sufficient to rescue cell proliferation in TAF6L knockdown organoids.

“Notably, the re-expression of TAF6L-GFP was sufficient to rescue cell proliferation in TAF6L knockdown organoids (Figs. 6E and S8D).”

GMDS OE organoids exhibited increased sensitivity to cisplatin treatment.

“Furthermore, GMDS expression effectively rescued the suppressed GMDS levels in cisplatin-treated organoids (Fig. S7E) and resulted in GMDS OE organoids exhibiting an increased sensitivity to cisplatin treatment (Fig. 5G), accompanied by an increase in nuclear phospho-H2AX (γH2AX) (Fig. 5H).”

Cisplatin, when combined with the knockdown of several NER genes, synergistically suppresses GMDS, leading to a marked reduction in total cellular fucosylated proteins.

“We found that cisplatin, when combined with the knockdown of several NER genes, synergistically suppresses GMDS, which encodes a rate-limiting enzyme in GDP-fucose biosynthesis, leading to a marked reduction in total cellular fucosylated proteins and suggesting a functional connection between DNA repair and fucosylation.”

• AssumptionRests on an assumption: GDP-fucose is the sole donor for protein fucosylation, a critical glycan modification for numerous membrane and secreted proteins.

Using iCRISPRi screens, we identified numerous hits already known to participate in DNA damage response to cisplatin treatment, such as genes involved in NER, HRR, and FA pathways.

“In line with this, using iCRISPRi screens, we identified numerous hits already known to participate in DNA damage response to cisplatin treatment, such as genes involved in nucleotide-excision repair (NER), homologous recombination repair (HRR), and Fanconi anemia (FA) pathways.”

Most genes with strong effects on cisplatin sensitivity were identified exclusively in the iCRISPRi screens, but not in the iCRISPRa screen.

“Interestingly, most genes with strong effects on cisplatin sensitivity were identified exclusively in the iCRISPRi screens, but not in the iCRISPRa screen.”

Pharmacological inhibition of fucosylation further sensitized cells to cisplatin.

“Supporting this hypothesis, pharmacological inhibition of fucosylation further sensitized cells to cisplatin, validating the functional relevance of this pathway in modulating drug response.”

The identification of biologically relevant hits, such as MYC, supports the potential utility of CRISPRa when cautiously interpreted.

“Nonetheless, the identification of biologically relevant hits, such as MYC, supports the potential utility of CRISPRa when cautiously interpreted.”

Our experiments across different organoid lines demonstrated that TAF6L expression can influence cellular sensitivity to cisplatin.

“Our experiments across different organoid lines demonstrated that TAF6L expression can influence cellular sensitivity to cisplatin.”

Our 3D CRISPR screens in human organoids revealed gene-specific cisplatin vulnerabilities that have not been detected in 2D genetic screens.

“Importantly, our 3D CRISPR screens in human organoids revealed gene-specific cisplatin vulnerabilities that have not been detected in 2D genetic screens.”

The results demonstrated the establishment of a large-scale CRISPR-based genetic screening platform in human 3D organoids.

“Overall, these results demonstrated the establishment of a large-scale CRISPR-based genetic screening platform in human 3D organoids, thereby increasing confidence in the identified target genes.”

The CRISPRi screens provide a strong capability to uncover genes associated with cisplatin sensitivity.

“These results suggested that the CRISPRi screens provide a strong capability to uncover genes associated with cisplatin sensitivity.”

Inhibition of RNA polymerase subunits such as POLR1D, POLR2B, and BRF2 may block sensing of DNA damage, resulting in cisplatin hypersensitivity.

“Moreover, consistent with the function of RNA polymerases as sensors of DNA damage-induced lesions45, inhibition of RNA polymerase subunits, such as POLR1D, POLR2B, and BRF2, may block sensing of DNA damage, resulting in cisplatin hypersensitivity.”

• cites [45] Lans, H., Hoeijmakers, J. H. J., Vermeulen, W. & Marteijn, J. A. The DNA damage response to transcription stress. Nat. Rev. Mol. Cell Biol. 20, 766–784 (2019). sourceNot checked — no full text of the source

The non-overlap of 3D organoid screen hits with previous 2D screens suggests potential 3D specificity.

“In particular, many genes identified from our iCRISPRi pooled screens in 3D human gastric organoids were not determined as cisplatin sensitization partners in previous genome-scale CRISPR KO screens in immortalized 2D human cell lines51, suggesting potential 3D specificity (Fig. S6A).”

TAF6L is critical for the proliferation that occurs during recovery following cisplatin treatment.

“These results suggested that TAF6L is critical for the proliferation that occurs during recovery following cisplatin treatment.”

Several key findings from the primary screens have been independently validated, reinforcing the credibility of the identified target genes.

“Notably, several key findings from our primary screens have been independently validated, further reinforcing the credibility of the identified target genes.”

The list of gene hits could be generally grouped into known biological processes related to cisplatin-associated DNA repair signaling based on published literature.

“With some exceptions, this intriguing list of gene hits could be generally grouped into known biological processes related to cisplatin-associated DNA repair signaling based on published literature (Fig. S6B).”

These results were independently validated in an additional TP53 KO organoid line.

“These results were independently validated in an additional TP53 KO organoid line (Fig. S8A).”

There is a strong autoregulatory effect at the TAF6L promoter.

“Of note, among the significantly reduced ATAC peaks in promoter regions, the peak located at the TAF6L promoter ranked as the 1st peak (Fig. S9D), consistent with a strong autoregulatory effect at the TAF6L promoter.”

These findings reveal a previously uncharacterized crosstalk between GMDS expression and cisplatin sensitivity.

“Taken together, these findings reveal a previously uncharacterized crosstalk between GMDS expression and cisplatin sensitivity, highlighting a potential mechanism by which cancer cells modulate their response to cisplatin.”

TAF6L does not directly regulate the expression of DNA repair genes or other related genes.

“We did not identify any significant GO terms related to the DNA damage response, suggesting that TAF6L does not directly regulate the expression of DNA repair genes or other related genes.”

GMDS downregulation is dissociated from cisplatin-induced cytotoxicity.

“This discrepancy thus dissociated GMDS downregulation from cisplatin-induced cytotoxicity and suggested that the synergistic suppression of GMDS observed with combined cisplatin treatment and NER gene knockdown was not inherently cytotoxic but rather reflected a positive feedback mechanism promoting cell survival following DNA damage.”

TAF6L depletion inhibited cell proliferation.

“However, TAF6L knockdown reduced the EdU+ cell population to an average of 8.0% compared to control organoids (18.1% EdU+ on average), indicating that TAF6L depletion inhibited cell proliferation.”

Other mechanisms may be at play for TAF6L-associated cisplatin sensitivity.

“This was consistent with the single-cell CRISPR results (Fig. 5), as TAF6L did not cluster with NER- or HRR-specific groups, indicating that other mechanisms may be at play.”

The synergistic suppression of GMDS observed with combined cisplatin treatment and NER gene knockdown was not inherently cytotoxic but reflected a positive feedback mechanism promoting cell survival following DNA damage.

“This discrepancy thus dissociated GMDS downregulation from cisplatin-induced cytotoxicity and suggested that the synergistic suppression of GMDS observed with combined cisplatin treatment and NER gene knockdown was not inherently cytotoxic but rather reflected a positive feedback mechanism promoting cell survival following DNA damage.”

TAF6L does not directly regulate DNA repair signaling activity.

“However, we did not observe significant changes in RAD51, FANCD2, or ubiquitinated FANCD2 (Ub-FANCD2) expression in TAF6L knockdown or TAG6L-GFP organoids (Fig. S8B, C), suggesting that TAF6L does not directly regulate DNA repair signaling activity.”

TAF6L may act as a cisplatin sensitization gene by maintaining global chromatin accessibility of loci required for proliferation recovery after chemotherapy exposure.

“Taken together, these results suggest that TAF6L may act as a cisplatin sensitization gene by maintaining global chromatin accessibility of loci required for proliferation recovery after chemotherapy exposure.”

The downregulation of GMDS-associated fucosylation functions as a DNA damage-induced pro-survival signal to reduce cisplatin sensitivity, independent of additional NER gene knockdown.

“These results supported a potential working model in which the downregulation of GMDS-associated fucosylation functions as a DNA damage-induced pro-survival signal to reduce cisplatin sensitivity, independent of additional NER gene knockdown (Fig. S7I).”

• AssumptionRests on an assumption: GDP-fucose is the sole donor for protein fucosylation, a critical glycan modification for numerous membrane and secreted proteins.

The cisplatin sensitization-induced transcriptomic harmonization reflected the known distinct biology of NER versus HRR DNA repair pathways.

“This cisplatin sensitization-induced transcriptomic harmonization faithfully reflected the known distinct biology of the NER versus HRR DNA repair pathways versus transcriptomic divergence observed upon iCRISPRi inhibition in cisplatin-naïve organoids.”

These known technical limitations likely contributed to the lower correlation observed in our CRISPRa replicates.

“These known technical limitations likely contributed to the lower correlation observed in our CRISPRa replicates.”

There is a functional connection between DNA repair and fucosylation.

“We found that cisplatin, when combined with the knockdown of several NER genes, synergistically suppresses GMDS, which encodes a rate-limiting enzyme in GDP-fucose biosynthesis, leading to a marked reduction in total cellular fucosylated proteins and suggesting a functional connection between DNA repair and fucosylation.”

3D culture systems may be particularly useful for uncovering cancer vulnerabilities.

“Recent studies have shown that phenotypes associated with CRISPR-mediated knockout of putative oncogenic loci are more pronounced when cancer cells are grown as 3D spheroids than 2D monolayers, suggesting that 3D culture systems may be particularly useful for uncovering cancer vulnerabilities55.”

More than 90% of sgRNA library-transduced cells became sgRNA positive after puromycin selection.

“More than 90% of sgRNA library-transduced cells became sgRNA positive after puromycin selection.”

Our data suggested that TAF6L may have additional functions beyond its role as a TATA-box binding protein-associated factor in regulating transcription machinery.

“Our data suggested that TAF6L may have additional, intriguing functions beyond its role as a TATA-box binding protein-associated factor in regulating transcription machinery.”

Genetic knockdown and overexpression of a given target may not produce reciprocal phenotypes due to saturation effects from homeostatic gene expression levels.

“This is consistent with the idea that genetic knockdown and overexpression of a given target may not produce reciprocal phenotypes due to saturation effects from homeostatic gene expression levels.”

Our single-cell CRISPR screens in 3D organoids uncovered a previously unrecognized link between cisplatin sensitivity and fucosylation.

“Beyond DNA repair, our single-cell CRISPR screens in 3D organoids further uncovered a previously unrecognized link between cisplatin sensitivity and fucosylation.”

GMDS/fucosylation suppression might serve a protective role in the context of cisplatin treatment.

“Interestingly, re-expressing GMDS did not rescue cells from cisplatin-induced cytotoxicity; instead, it enhanced cell death, indicating that GMDS/fucosylation suppression might serve a protective role in the context of cisplatin treatment.”

These unexpected results suggest a potential crosstalk between DNA repair and fucosylation.

“These unexpected results suggest a potential crosstalk between DNA repair and fucosylation, highlighting the need for further investigation into its mechanistic basis.”

The gene-specific dependencies and biological processes identified may be distinct to early cancer development as opposed to established tumors.

“Finally, our oncogene-engineered human tumor organoids represent a pre-cancerous stage9,37, meaning the gene-specific dependencies and biological processes identified may be distinct to early cancer development as opposed to established tumors.”