Development of a Mesophyll Protoplast-Based System for Gene Editing of Papaya

Carica papaya L. is an economically significant crop in tropical and subtropical regions, with a gross production value of $6.2 × 10 9 in 2020. However, various biotic and abiotic stresses threaten crop productivity. To enhance stress resistance, genetic engineering and traditional breeding have been employed. Unfortunately, these methods are limited by the scarcity of innate disease resistance genes in the genome and the poor fertility of interspecific hybrids. Therefore, to circumvent these limitations, we developed a papaya protoplast-based gene editing system. By optimizing protoplast isolation, 28% higher yields were achieved from older (≥75 d) plants at 1.11 × 10 8 ± 0.069 protoplasts per gram-fresh-weight. Protoplast viability was 89.87 ± 2.02%,. We established an efficient genetic transfection method and verified proper expression, cellular function and localization of GFP and PDI-mCherry fusions in the protoplasts. Using preassembled CRISPR-Cas9 ribonucleoprotein complexes, we successfully edited a mutant GFP transgene, resulting in a frame-shift restoration efficiency of 27.88 ± 1.65%. Next, the CpPDS and CpMLO6 genes were targeted, creating knockouts in three different papaya cultivars. The average CpPDS mutant frequency obtained was 42.31 ± 1.90%, of which 31.25 ± 1.46% were frame-shift knockouts, while 11.05 ± 1.37% were in-frame protein variants. The average CpMLO6 mutant frequency was 16.20 ± 1.53%, of which 13.71 ± 1.67% were frame-shift knockouts and 2.50 ± 0.26% were in-frame variants. Taken together, a DNA-free CRISPR-Cas9 gene editing system was successfully demonstrated in papaya protoplasts on multiple target genes for use in papaya crop improvement.