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

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Abstract

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.

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Acknowledgements

We thank Dr. Bing Yang for the GFPm construct and advice on its use, and Mrs. Tina Weatherby for expert advice on microscopy.

Dr. Bing Yang

Bond Life Sciences Center

Division of Plant Sciences

University of Missouri

1201 Rollins Street, Columbia, MO 65211

Tina Weatherby

Biological Electron Microscope Facility

Life Sciences Building

University of Hawai’i at Mānoa

1800 East-West Road, Honolulu, HI 96822

Funding

This work was supported by the University of Hawai’i at Mānoa and the USDA-NIFA-AFRI award 2020-67013-31549.

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Authors and Affiliations

  1. Department of Molecular Biosciences and Bioengineering, University of Hawai’i at Mānoa, 1955 East-West Road, Honolulu, HI, 96822, USA

    Marc J. Elias & David A. Christopher

  2. Department of Plant and Environmental Protection Sciences, University of Hawai’i at Mānoa, 3190 Maile Way, Honolulu, HI, 96822, USA

    Jeremieh Hasley & Miaoying Tian

Authors
  1. Marc J. Elias
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  2. Jeremieh Hasley
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  3. Miaoying Tian
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  4. David A. Christopher
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Corresponding author

Correspondence to David A. Christopher.

Additional information

Responsible Editor: Yong Eui Choi

Supplementary information

ESM 1

Supplemental file 1: Phylogenetic analysis of the candidate S-gene MLO-like protein homologs in papaya. (PDF 392 kb)

ESM 2

Supplemental file 2: The secondary structure of selected sgRNA candidates targeting Carica papaya endogenous genes, CpPDS and CpMLO6. (PDF 379 kb)

ESM 3

Supplemental file 3: In vitro DNA cleavage activity of CpPDS-sgRNA16 and CpMLO6-sgRNA254. (PDF 247 kb)

ESM 4

Supplemental file 4: Table 1: Indels generated during initial DNA-free CRISPR-Cas9 targeted gene editing of CpPDS in papaya cv. Solo Sunrise. Table 2: Indels generated during replicate DNA-free CRISPR-Cas9 targeted gene editing of CpPDS in papaya cv. Solo Sunrise. Table 3: Indels generated during DNA-free CRISPR-Cas9 targeted gene editing of CpPDS in papaya cv. Solo Sunset. Table 4: Indels generated during DNA-free CRISPR-Cas9 targeted gene editing of CpPDS in papaya cv. Solo Kapoho. (PDF 2013 kb)

ESM 5

Supplemental file 5: Summary of DNA-free CRISPR-Cas9 targeted gene editing of CpPDS and CpMLO6 in papaya cultivars Solo Sunrise, Sunset, and Kapoho. (PDF 206 kb)

ESM 6

Supplemental file 6: Table 5: Indels generated during initial DNA-free CRISPR-Cas9 targeted gene editing of CpMLO6 in papaya cv. Solo Sunrise. Table 6: Indels generated during replicate DNA-free CRISPR-Cas9 targeted gene editing of CpMLO6 in papaya cv. Solo Sunrise. Table 7: Indels generated during DNA-free CRISPR-Cas9 targeted gene editing of CpMLO6 in papaya cv. Solo Sunset. Table 8: Indels generated during DNA-free CRISPR-Cas9 targeted gene editing of CpMLO6 in papaya cv. Solo Kapoho. (PDF 1700 kb)

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Elias, M.J., Hasley, J., Tian, M. et al. Development of a Mesophyll Protoplast-Based System for Gene Editing of Papaya. In Vitro Cell.Dev.Biol.-Plant 59, 517–535 (2023). https://doi.org/10.1007/s11627-023-10373-1

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