Allele-specific disruption of KRAS p.G12V in colorectal cancer cells using electroporated Cas9 RNPs

Abstract

Objective(s): Colorectal cancer (CRC) is a major cause of mortality, and KRAS mutations drive tumorigenesis and therapy resistance. Although CRISPR-Cas9 enables precise genome editing, intracellular delivery of Cas9 ribonucleoproteins (RNPs) remains challenging. We evaluated direct electroporation of Cas9-sgRNA RNPs targeting KRAS p.G12V in SW480 CRC cells to assess delivery efficiency, on-target editing, off-target activity, and viability effects. Materials and Methods: An sgRNA targeting KRAS p.G12V was designed computationally. Cas9-sgRNA RNPs were assembled and validated by in vitro digestion. RNPs were electroporated into SW480 cells using a square-wave electroporation protocol; pmaxGFP served as the delivery control. On-target editing and five predicted off-target loci were assessed by Sanger sequencing and TIDE analysis. Cell viability was measured by the MTT assay. Results: Electroporation resulted in high GFP expression (~97%). TIDE analysis at the KRAS locus estimated ~27% total indels. Cells treated with KRAS-targeting RNPs exhibited significantly reduced viability compared with controls (P<0.05). No detectable indels were observed at the five off-target sites. While supporting feasibility, deeper sequencing and mechanistic analyses are needed to confirm specificity. Conclusion: Direct electroporation of Cas9-sgRNA RNPs enabled efficient intracellular delivery and targeted disruption in SW480 cells, reducing viability without detectable editing at selected off-target loci. This approach is practical for KRAS-targeted studies in CRC and warrants further investigation. Direct electroporation of Cas9--sgRNA RNPs enabled efficient delivery in SW480 cells, accompanied by reduced viability, with no detectable editing at selected off-target candidates by Sanger/TIDE. These findings support RNP electroporation as a practical approach for KRAS-targeted studies in CRC and justify follow-up with amplicon deep sequencing and additional cell models.