CRISPR/Cas12a-mediated allele engineering of SmAPRR2 and SmGLK2 reveals complementary control of fruit peel and flesh chlorophyll pigmentation in eggplant

Eggplant (Solanum melongena L.) displays extensive fruit color diversity, in which chlorophyll-related pigmentation contributes to both external appearance and market value. Previous genetic studies identified SmAPRR2 and SmGLK2 as major candidate genes controlling uniform green pigmentation and green netting in fruit, respectively, but their individual and combined functional contributions had not been validated through targeted mutagenesis in a common genetic background. Here, we established a multiplex CRISPR/Cas12a system in eggplant accession MEL3, representing, to our knowledge, the first application of this nuclease for genome editing in eggplant. Transformation efficiency was 2.0%, but all 15 genotyped regenerants were edited, yielding four SmAPRR2 and six SmGLK2 alleles. Segregation and crossing enabled the recovery of six transgene-free lines carrying single or combined edited alleles. Disruption of SmGLK2 abolished the reticulated green netting pattern while preserving a uniformly green peel and the internal green ring. Conversely, disruption of SmAPRR2 reduced the background uniform peel pigmentation and eliminated the green ring while retaining green netting. Double mutants carrying disruptive alleles at both loci produced white fruits lacking internal green pigmentation, whereas putatively hypomorphic SmAPRR2 and SmGLK2 alleles generated intermediate phenotypes. Whole-genome resequencing identified only two predicted off-target sites under a canonical TTTV PAM search allowing up to four mismatches. Both were fully covered, and no edited-line-specific candidate variants were detected. These findings establish complementary and partially separable roles for SmAPRR2 and SmGLK2 in fruit peel and flesh chlorophyll pigmentation and demonstrate the potential of Cas12a for functional genomics, allele engineering, and precision breeding in eggplant.