Highlight: HiFiCas9 Selectively Disables Mutant KRAS in Lung Tumours

Comprehensive specificity validation demonstrated selective editing only in cell lines harbouring the corresponding mutations, with no detectable activity against wild-type KRAS(KRAS^WT) alleles. Next-generation sequencing confirmed mutation-specific targeting, revealing editing frequencies of 64.7% and 78.2% for G12C and G12D, respectively, while KRAS^WT alleles showed editing frequencies below 2.1% (see Figure 1F).

»Instead of blocking the mutant protein, we aimed to eliminate the mutation directly at the DNA level. Using HiFi-Cas9, we designed guides that specifically recognise the two most common KRAS mutations in lung cancer, cutting only the mutant DNA while leaving the healthy copy intact,« Pedro Medina explains to CRISPR Medicine News. He is professor at the University of Granada and senior author of the paper that was published Monday in Nature Communications.

Functional validation revealed significant cytotoxic effects in KRAS-mutant cell lines. Ribonucleoprotein delivery produced viability reductions of 67% (H358, G12C), 26% (H1792, G12C), 36% (A427, G12D) and 32% (SK-LU-1, G12D) at seven days post-treatment, whilst KRAS^WT H838 cells showed no significant reduction. Effects were enhanced in three-dimensional spheroid cultures, consistent with increased KRAS dependency in physiologically relevant conditions.

Comparative analysis with sotorasib, the FDA-approved G12C covalent inhibitor, revealed superior HiFiCas9 performance. Two-way ANOVA analysis indicated that HiFiCas9 accounted for 72.96% of total variance, whilst sotorasib contributed only 13.3%. Combination treatment provided no additional benefit, consistent with non-overlapping mechanisms – genetic elimination versus pharmacological inhibition of GDP-bound protein.

Patient-derived xenograft organoid studies using three models revealed heterogeneous responses reflecting clinical diversity. TP79 responded exclusively to HiFiCas9, with no sensitivity to sotorasib, while TP60 responded to both treatments.

These findings highlight potential therapeutic advantages in sotorasib-refractory cases where high GTP-bound G12C levels may drive resistance.

»In preclinical models, HiFi-Cas9 slowed tumour growth and even outperformed sotorasib in some cases, including resistant tumours. This suggests that CRISPR-based strategies can address vulnerabilities beyond the reach of current drugs and opens the way towards more durable therapies once in vivo delivery is optimised,« Pedro Medina says.

Evaluation of sotorasib-resistant cell lines demonstrated context-dependent efficacy. H23 resistant cells, harbouring co-occurring KEAP1 and SMARCA4 mutations associated with poor clinical outcomes, showed significant viability reduction (60%) in 3D cultures. Sequential treatment experiments revealed no evidence of adaptive resistance development across three consecutive treatments, suggesting durable therapeutic potential.

The study demonstrates that carefully engineered HiFiCas9 systems can achieve clinically relevant single-nucleotide discrimination for oncogene targeting. The superior performance compared to current G12C inhibitors, combined with activity against G12D (for which no approved therapies exist), establishes this approach as a potentially transformative therapeutic strategy requiring advances in in vivo delivery optimisation.

This study was conducted by Pedro Medina, Juan Carlos Álvarez-Pérez, Juan Sanjuán-Hidalgo, Alberto M. Arenas and colleagues from the University of Granada, Spain, in collaboration with collaborators from multiple Spanish institutions. The study was published in Nature Communications on 1 September 2025.