Using a CRISPR-Cas Whole-Genome Screen to Identify Genetic Factors Linked to expression of Cancer-Associated O-glycans
Bibliographic record
Abstract
Truncated O-glycans, designated Tn and STn, have been recognized as cancer-associated antigens correlated with worsened patient outcomes for more than four decades. In epithelial cancers, truncated O-glycans are identified in 70-90 % of cases and are often seen in premalignant lesions, making them attractive therapeutic targets as well as interesting biological entities potentially linked to fundamentally important processes in carcinogenesis. However, little is still known of the exact mechanism(s) resulting in the expression of truncated O-glycans in cancer cells. Several hypotheses have been proposed, including mutations of the CORE1 and COSMC genes, pH-dependent regulation, methylation of gene promotors, relocation of GalNAc-transferases in the secretory pathway and substrate deficiencies, but none of these hypotheses have yet to explain more than a limited subset of cases. To investigate potential genetic factors linked to Tn and STn expression, we therefore conducted a whole-genome genetic perturbation screen in the human keratinocyte HaCaT cell line with stable expression of the Cas9-protein and no expression of truncated O-glycans. Employing the glycan-specific monoclonal antibodies to Tn (mAb 5F4) and STn (mAb 3F1), we subsequently used FACS to separate cells with perturbated genes resulting in Tn or STn expression from keratinocytes not expressing truncated O-glycans. We found a subset (approximately 0.05 %) of the genetically modified cells from the whole-genome screen to be Tn- or STn-positive when analyzed på flow cytometry, with a larger proportion of Tn-positive cells as compared to STn-positive cells. We are currently using Next Generation Sequencing (NGS) on PCR products amplified from the signature sgRNA sequences imbedded in the lentiviral inserts from the Brunello library to identify candidate genes which – when knocked out – results in expression of truncated O-glycans in the Tn- and STn-positive cell populations. We aim to validate the candidate genes by conducting single-gene knock outs of the genes targeted by the most abundant sgRNAs in the Tn- and STn-positive cell populations. Subsequently, we seek to evaluate the single-cell knock outs by antibody staining, lectin staining and MS/MS to confirm the expression of Tn- and STn-antigens, as implied by the findings in the whole-genome screen. In conclusion, we conducted a whole-genome screen investigating the effect on expression of truncated O-glycans from perturbation of single genes by CRISPR-Cas9 induced knock outs in a human cell line. We find that the whole-genome screen results in expression of Tn- and STn-expression in a small number of cells – but a larger number than what can be explained from COSMC and CORE1 perturbations exclusively – and aim to present candidate genes and validated genetic targets involved in expression of truncated O-glycans in a human setting.
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How this classification was reachedexpand
Full frame machine prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. The Gemma side is a direct model label for every work in the frame, read from the title-only record. The Codex side is a classifier learned from the 10,348 direct Codex labels and calibrated to design-weighted sample rates; fields without enough sample support carry no Codex call. Candidate is the union of the two sides; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels.
Distilled classifier scores by category (both heads)
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.000 | 0.000 |
| Meta-epidemiology (narrow) | 0.001 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.000 |
| Bibliometrics | 0.000 | 0.000 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.001 | 0.001 |
| Insufficient payload (model declined to judge) | 0.001 | 0.000 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one source (direct Gemma or distilled Codex), not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".