CRISPR-Cas9 Knockout of KRAS-G12C Effector RAF1 in NCI-H358 Lung Adenocarcinoma Cells with Phospho-MEK Validation

Two RNP conditions—RAF1-targeting sgRNA and a non-targeting control sgRNA—each electroporated in n=3 independent transfections. From the RAF1-edited pool, ≥12 single-cell clones are isolated and screened; ≥3 confirmed biallelic-knockout clones and ≥3 control clones are carried to phenotyping to control for clonal variation. Phospho-MEK immunoblot is run with the analyst blinded to clone genotype. Two independent sgRNAs targeting different RAF1 exons are used to control for off-target effects.

BSL-2 for NCI-H358 human tumor cells; biosafety cabinet for all open work. Cas9 RNP electroporation is non-viral and low-biohazard (no infectious vector), but follow institutional IBC approval for gene editing. Electroporation cuvettes carry high-voltage—follow device interlocks. RIPA/methanol and ECL handled with gloves/eye protection. Decontaminate cell waste in 10% bleach 30 min; autoclave solid biohazard waste. Maintain phospho-protein cold chain during lysis.

Non-targeting sgRNA RNP clones = genotype-matched negative control (full editing/cloning workflow, no RAF1 disruption). Parental unedited NCI-H358 = baseline. Positive editing control = a previously validated sgRNA against a housekeeping locus to confirm electroporation efficiency. Two independent RAF1 sgRNAs control for off-target phenotypes (concordant phospho-MEK reduction supports on-target effect). RAF1 immunoblot is the direct knockout-confirmation control.

ICE/TIDE shows >70% indels in the edited pool. ≥25% of screened clones are biallelic frameshift KO with undetectable RAF1 protein. KO clones show 30-60% lower normalized p-MEK1/2 than control clones, with unchanged total MEK1/2 and vinculin. Concordant reduction across two independent sgRNAs and across ≥3 clones supports an on-target, reproducible phenotype.

• NCI-H358 cells (ATCC CRL-5807), KRAS-G12C lung adenocarcinoma
• RPMI-1640 + 10% FBS + 1% pen/strep
• Recombinant Cas9 nuclease (NLS), 20 µM
• Synthetic sgRNA targeting RAF1 exon 3 (and a second exon as backup), 100 µM
• Non-targeting control sgRNA
• Electroporation buffer + nucleofection device cuvettes
• Genomic DNA extraction kit; RAF1 flanking PCR primers; ICE/TIDE analysis
• Anti-RAF1 (c-Raf) rabbit mAb, 1:1000
• Anti-phospho-MEK1/2 (Ser217/221) (41G9) rabbit mAb, 1:1000
• Anti-MEK1/2 (47E6) rabbit mAb, 1:1000
• Anti-vinculin loading control, 1:5000
• RIPA + Halt protease/phosphatase inhibitors; 4-12% Bis-Tris gels; PVDF; ECL

To produce and validate clonal RAF1-knockout NCI-H358 cells using a Cas9 RNP electroporation approach (transient, plasmid-free, low off-target), confirm editing by Sanger sequencing/ICE analysis and immunoblot, and quantify the downstream consequence on phospho-MEK1/2 to interrogate RAF1's role in KRAS-G12C effector signaling.

1. Assemble RNP: incubate 100 pmol Cas9 + 120 pmol sgRNA in electroporation buffer, 15 min RT (1.2:1 sgRNA:Cas9).
2. Resuspend 2 x 10^5 NCI-H358 cells in buffer; combine with RNP; electroporate per device program for adherent lung lines.
3. Recover cells in pre-warmed antibiotic-free RPMI in a 24-well plate, 48-72 h.
4. Harvest a pool aliquot; extract gDNA, PCR the RAF1 locus, Sanger sequence, and quantify indels by ICE/TIDE.
5. Limiting-dilution single-cell clone the edited pool into 96-well plates (0.5 cells/well); expand 2-3 weeks.
6. Screen clones by RAF1-locus PCR + sequencing for biallelic frameshift; confirm protein loss by anti-RAF1 immunoblot.
7. Expand ≥3 KO and ≥3 control clones; lyse subconfluent cultures in RIPA + inhibitors.
8. Run 20 µg/lane on 4-12% gel; transfer to PVDF.
9. Probe p-MEK1/2, then total MEK1/2, RAF1, and vinculin.
10. Image by ECL; quantify p-MEK/total-MEK normalized to vinculin.

Independent variable: RAF1 genotype (knockout vs non-targeting control). Dependent variable: phospho-MEK1/2 (Ser217/221) normalized to total MEK1/2 and vinculin. Controlled variables: cell passage, confluence at lysis (subconfluent, ~70%), RNP composition and dose, clonal expansion conditions, lysis/loading (20 µg), and antibody lots. Editing efficiency and clonality are confirmed, not assumed.

We hypothesize that RAF1 contributes to mutant-KRAS-driven MEK activation in NCI-H358 cells, such that biallelic RAF1 knockout reduces basal phospho-MEK1/2 by 30-60% relative to a non-targeting control clone, without abolishing it (due to BRAF/ARAF redundancy), while total MEK1/2 remains unchanged.

Quantify indel spectra with ICE/TIDE from Sanger traces. Densitometry (ImageJ/Fiji) of p-MEK, total MEK, RAF1, and vinculin; compute p-MEK/total-MEK normalized to vinculin per clone. Average technical immunoblot replicates per clone, then treat each clone as a biological unit. Compare KO vs control clone groups; present per-clone dot plots overlaid on group means to expose clonal variability.

1. Low editing efficiency (ICE <30%): re-optimize RNP ratio and electroporation program; verify sgRNA quality and Cas9 activity with a control locus. 2. No clones grow: NCI-H358 single-cell cloning is inefficient—use conditioned medium or feeder support and seed multiple plates. 3. Mixed/heterozygous clones: re-screen and sub-clone; require sequencing-confirmed biallelic frameshift before phenotyping. 4. No p-MEK change despite KO: expected if BRAF/ARAF compensate—report as redundancy; consider double knockdown. 5. Off-target concern: confirm phenotype with the second independent sgRNA before drawing conclusions.

Clones are the experimental unit: ≥3 KO vs ≥3 control clones. Compare normalized p-MEK by a two-tailed unpaired t-test (or Mann-Whitney if non-normal), alpha = 0.05; a nested/mixed model is preferred if multiple lysates per clone are used to avoid pseudoreplication. Report effect size (Cohen's d) and exact p-values. Concordance across two sgRNAs is reported descriptively. Power: detecting a 40% mean reduction (d≈2 across clones) gives >0.8 power at n=3 per group.