Saturation Genome Editing Targeting KRAS Mutations in HCT 116 Colon Carcinoma Cells for Pooled SNV Functional Profiling in Diploid Cancer Model
Abstract
1. Introduction
2. Materials and Methods
2.1. Cell Culture
2.2. Saturation Genome Editing
2.3. Massive Parallel Sequencing
2.4. Single Nucleotide Variant Analysis
3. Results
3.1. Saturation Genome Editing of KRAS
3.2. KRAS G12/G13 Mutations Enhanced Cell Growth
3.3. Drug Sensitivity Test
4. Discussion
5. Conclusions
6. Limitations
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
Abbreviations
| Cas9 | Clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 |
| FDR | False discovery rate |
| GOI | Gene of interest |
| HDR | Homology-directed repair |
| LFC | Log2 fold change |
| mES | Mouse embryonal stem cell |
| OG | Oncogene |
| PAM | Protospacer adjacent motif |
| PCR | Polymerase chain reaction |
| RNP | Ribonucleoprotein |
| SGE | Saturation genome editing |
| sgRNA | Single-guide RNA |
| SNV | Single-nucleotide variant |
| ssODN | Single-strand oligodeoxynucleotide |
| TSG | Tumor suppressor gene |
| VUS | Variants of uncertain significance |
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| # Reference (Year) | GOI | Class of GOI | # SNVs | Delivery | Cell | |
|---|---|---|---|---|---|---|
| Cas9 | Donors | |||||
| [4] (2014) | BRCA1/DBR1 | TSG/- | 234/225 | plasmid | plasmid | HAP1 |
| [5] (2018) | BRCA1 | TSG | 3893 | plasmid | ssODN | HAP1 |
| [9] (2020) | CARD11 | OG | 2542 | RNP | plasmid | TMD8 |
| [6] (2023) | DDX3X | TSG | 12776 | plasmid | plasmid | HAP1 |
| [20] (2023) | BRCA2 | TSG | 851 | plasmid | ssODN | mES |
| [21] (2024) | RAD51C | TSG | 9188 | HAP1-A5 | plasmid | HAP1 |
| [7] (2024) | VHL | TSG | 2268 | plasmid | plasmid | HAP1 |
| [22] (2024) | BAP1 | TSG | 18108 | HAP1-A5 | plasmid | HAP1 |
| [10] (2025) | TP53 | TSG | 9225 | plasmid | plasmid | HCT 116* |
| [23] (2025) | BRCA2 | TSG | 6959 | plasmid | plasmid | HAP1 |
| [24] (2025) | BRCA2 | TSG | 6551 | plasmid | plasmid | mES |
| present study | KRAS | OG | 36 | RNP | ssODN | HCT 116 |
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Hamada, T.; Yokoyama, S.; Nakabayashi, R.; Suzuki, Y.; Morishita, S.; Akahane, T.; Matsuo, K.; Kitazono, I.; Furukawa, T.; Tanimoto, A. Saturation Genome Editing Targeting KRAS Mutations in HCT 116 Colon Carcinoma Cells for Pooled SNV Functional Profiling in Diploid Cancer Model. Curr. Issues Mol. Biol. 2026, 48, 341. https://doi.org/10.3390/cimb48040341
Hamada T, Yokoyama S, Nakabayashi R, Suzuki Y, Morishita S, Akahane T, Matsuo K, Kitazono I, Furukawa T, Tanimoto A. Saturation Genome Editing Targeting KRAS Mutations in HCT 116 Colon Carcinoma Cells for Pooled SNV Functional Profiling in Diploid Cancer Model. Current Issues in Molecular Biology. 2026; 48(4):341. https://doi.org/10.3390/cimb48040341
Chicago/Turabian StyleHamada, Taiji, Seiya Yokoyama, Ryo Nakabayashi, Yoshihiko Suzuki, Shinichi Morishita, Toshiaki Akahane, Kei Matsuo, Ikumi Kitazono, Tatsuhiko Furukawa, and Akihide Tanimoto. 2026. "Saturation Genome Editing Targeting KRAS Mutations in HCT 116 Colon Carcinoma Cells for Pooled SNV Functional Profiling in Diploid Cancer Model" Current Issues in Molecular Biology 48, no. 4: 341. https://doi.org/10.3390/cimb48040341
APA StyleHamada, T., Yokoyama, S., Nakabayashi, R., Suzuki, Y., Morishita, S., Akahane, T., Matsuo, K., Kitazono, I., Furukawa, T., & Tanimoto, A. (2026). Saturation Genome Editing Targeting KRAS Mutations in HCT 116 Colon Carcinoma Cells for Pooled SNV Functional Profiling in Diploid Cancer Model. Current Issues in Molecular Biology, 48(4), 341. https://doi.org/10.3390/cimb48040341

