Carica papaya L. is an economically significant crop in tropical and subtropical regions, with a gross production value of $6.2 × 10⁹ 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 × 10⁸ ± 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.

番木瓜是热带和亚热带地区的一种重要经济作物， 2020 年总产值为 6.2 × 10 ^{9美元。然而，各种生物和非生物胁迫威胁着作物生产力。}为了增强抗逆性，已采用基因工程和传统育种。不幸的是，这些方法受到基因组中先天抗病基因的缺乏和种间杂种的繁殖力差的限制。因此，为了规避这些限制，我们开发了一种基于木瓜原生质体的基因编辑系统。^{通过优化原生质体分离，1.11 × 10 8}的老植物（≥75 天）的产量提高了 28%± 0.069 个原生质体每克鲜重。原生质体活力为89.87±2.02%。我们建立了一种有效的遗传转染方法，并验证了 GFP 和 PDI-mCherry 融合体在原生质体中的正确表达、细胞功能和定位。使用预组装的 CRISPR-Cas9 核糖核蛋白复合物，我们成功编辑了突变型 GFP 转基因，移码恢复效率达到 27.88 ± 1.65%。接下来，CpPDS和CpMLO6基因被定位，在三个不同的木瓜品种中进行敲除。获得的CpPDS突变频率平均为42.31±1.90%，其中31.25±1.46%为移码敲除，11.05±1.37%为框内蛋白变异。平均CpMLO6突变频率为16.20±1.53%，其中13.71±1.67%为移码敲除，2.50±0.26%为框内变异。总之，无 DNA CRISPR-Cas9 基因编辑系统在木瓜原生质体中成功演示了多个靶基因，可用于木瓜作物改良。