Saturation Genome Editing Targeting KRAS Mutations in HCT 116 Colon Carcinoma Cells for Pooled SNV Functional...

Area of Science:

• Genetics
• Cancer Biology
• Molecular Biology

Background:

• Evaluating cancer gene mutations is crucial for targeted therapy selection.
• Massive parallel sequencing often identifies variants of uncertain significance (VUS).
• Saturation genome editing (SGE) using CRISPR-Cas9 aids VUS analysis by introducing numerous mutations.

Purpose of the Study:

• To develop and validate an SGE-based system for KRAS mutation analysis in diploid HCT 116 colon carcinoma cells.
• To assess the applicability of SGE in diploid cells beyond haploid HAP1 cells.
• To evaluate drug sensitivity using SGE in a cancer cell model.

Main Methods:

• Developed a Cas9-based SGE system for diploid HCT 116 cells.
• Generated single-nucleotide variants (SNVs) in KRAS codons A11-V14.
• Utilized massive parallel sequencing to analyze SNV abundance and drug response.

Main Results:

• Successfully generated KRAS SNVs in diploid HCT 116 cells.
• Observed changes in KRAS SNV abundance (e.g., G12, G13, G12C) after treatment with a KRAS inhibitor.
• Demonstrated SGE's utility in assessing drug sensitivity and SNV population dynamics.

Conclusions:

• SGE is applicable to diploid HCT 116 colon carcinoma cells, expanding its use beyond haploid models.
• The developed SGE system provides a practical framework for studying KRAS mutations in relevant cancer cells.
• This approach facilitates the evaluation of SNV population changes and drug sensitivity in cancer research.