CRISPR/Cas9-Based Knock-Out of the PMR4 Gene Reduces Susceptibility to Late Blight in Two Tomato Cultivars
Abstract
:1. Introduction
2. Results
2.1. CRISPR/Cas9-Based Mutagenesis of SlPMR4
2.2. Reduced Susceptibility to Late Blight (LB)
2.3. Genomics of Selected Pmr4 Mutants
2.4. Off-Target Events and SNP Analysis
3. Discussion
3.1. Reduced Susceptibility to LB in Tomato Cultivars Knocked-Out in the SlPMR4 Gene
3.2. Genome Editing Outcomes in Tomato Cultivars
3.3. Off-Target Absence and Emerging SNPs in Pmr4 Mutants
4. Materials and Methods
4.1. Plant Material and Transformation Vector
4.2. Plant Transformation, Regeneration, and Acclimation to Soil
4.3. Identification of Transformed Plants and Detection of the SlPMR4 Editing
4.4. Detached Leaf Assay with Phytophthora Infestans
4.5. Whole-Genome Sequencing
4.6. On- and Off-Target Analyses and SNP Statistics
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Mutant | Mutational Status (%)-TIDE | |||
---|---|---|---|---|
sgRNA6 | sgRNA8 | sgRNA1 | sgRNA7 | |
SM1 | 1.2 | - | - | - |
SM2 | 2.5 | - | - | - |
SM3 | 8.7 | - | - | - |
SM4 | 38.5 | 89.4 | - | 98 |
SM5 | 24.2 | - | - | 97.6 |
SM6 | 40.2 | 95.1 | 4.1 | 98.9 |
SM7 | 13.1 | 94.5 | 58.1 | - |
SM8 | 34.6 | 95.5 | 33.6 | - |
SM9 | - | 95.4 | 35.3 | - |
SM12 | - | 93.3 | 9.1 | - |
SM13 | 93.4 | 90.3 | 48.1 | 99 |
SM14 | - | 92.8 | 3.4 | - |
SM16 | - | 97.8 | 7.6 | - |
SM17 | 64.6 | 94.2 | 76.2 | 97.9 |
SM18 | - | 92.9 | 80.1 | - |
SM19 | 1.5 | 93.1 | 31.3 | 99.3 |
SM20 | 1.8 | 1.1 | 3.7 | 2.2 |
SM22 | 69.3 | 94.2 | 9.7 | 98.3 |
SM24 | - | 93.6 | 34.2 | - |
SM25 | - | 94.2 | 5.2 | - |
SM26 | - | 93.9 | 4.4 | - |
OX1 | 55.3 | - | - | - |
OX2 | 17.7 | 98.6 | 6.6 | 99 |
OX3 | 26.8 | 98.2 | 56.7 | 99.3 |
OX4 | 14.2 | 99 | 9.9 | 99 |
OX9 | 13.5 | 95.9 | 4.7 | 99.3 |
OX11 | 48.6 | 94.4 | 32.5 | 99.3 |
Mutant | Alleles in sgRNA7 Region (%) | |||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
R2 | −18 | −10 | −8 | −7 | −6 | −5 | −3 | −2 | −1 | 1 | ref | Allelic State | ||
SM6 | WGS | - | - | - | - | 100.0 | - | - | - | - | - | - | - | homozygous |
Tide | 0.99 | - | - | - | 98.9 | - | - | - | - | - | - | - | ||
SM13 | WGS | - | - | - | - | 100.0 | - | - | - | - | - | - | - | homozygous |
Tide | 0.98 | - | - | - | 98.5 | - | - | - | - | - | - | - | ||
SM17 | WGS | - | - | - | - | 100.0 | - | - | - | - | - | - | - | homozygous |
Tide | 0.98 | - | - | - | 97.8 | - | - | - | - | - | - | - | ||
SM19 | WGS | - | - | - | - | 100.0 | - | - | - | - | - | - | - | homozygous |
Tide | 0.99 | - | - | - | 97.2 | - | - | - | - | - | - | - | ||
SM-CTRL | WGS | - | - | - | - | - | - | - | - | - | - | - | 100.0 | wild type |
Mutant | Alleles in sgRNA1 Region (%) | |||||||||||||
R2 | −18 | −10 | −8 | −7 | −6 | −5 | −3 | −2 | −1 | 1 | ref | allelic state | ||
SM6 | WGS | - | - | 10.0 | - | - | - | - | - | - | 23.3 | 69.0 | chimeric | |
Tide | 0.96 | - | - | - | - | - | - | - | - | - | - | 92.2 | ||
SM13 | WGS | - | - | - | - | - | 52.4 | - | - | - | - | - | 47.6 | heterozygous |
Tide | 0.92 | - | - | - | - | 45.7 | - | - | - | - | - | 43.9 | ||
SM17 | WGS | - | - | - | - | - | - | - | - | 62.5 | 8.3 | 8.3 | 20.8 | chimeric |
Tide | 0.94 | - | - | - | - | - | - | - | 54.7 | 15.0 | 3.2 | 18.3 | ||
SM19 | WGS | - | - | - | 43.7 | - | - | - | - | - | - | - | 56.3 | heterozygous |
Tide | 0.91 | - | - | 27.6 | - | - | - | - | - | - | - | 60.0 | ||
SM-CTRL | WGS | - | - | - | - | - | - | - | - | - | - | - | 100.0 | wild type |
Mutant | Alleles in sgRNA8 Region (%) | |||||||||||||
R2 | −18 | −10 | −8 | −7 | −6 | −5 | −3 | −2 | −1 | 1 | ref | allelic state | ||
SM6 | WGS | - | - | - | - | 33.3 | - | - | - | 66.7 | - | - | - | biallelic |
Tide | 0.95 | - | - | - | 46.4 | - | - | - | 48.3 | - | - | - | ||
SM13 | WGS | - | - | - | - | 66.7 | - | - | - | 33.3 | - | - | - | biallelic |
Tide | 0.9 | - | - | - | 43.4 | - | - | - | 45.8 | - | - | - | ||
SM17 | WGS | - | - | - | - | 20.0 | - | - | - | 80.0 | - | - | - | biallelic |
Tide | 0.94 | - | - | - | 43.2 | - | - | - | 50.1 | - | - | - | ||
SM19 | WGS | - | - | - | - | 50.0 | - | - | - | 50.0 | - | - | - | biallelic |
Tide | 0.93 | - | - | - | 45.1 | - | - | - | 47.7 | - | - | - | ||
SM-CTRL | WGS | - | - | - | - | - | - | - | - | - | - | - | 100.0 | wild type |
Mutant | Alleles in sgRNA6 Region (%) | |||||||||||||
R2 | −18 | −10 | −8 | −7 | −6 | −5 | −3 | −2 | −1 | 1 | ref | allelic state | ||
SM6 | WGS | - | - | - | - | 5.7 | - | 14.3 | - | 17.1 | - | 45.7 | 17.1 | chimeric |
Tide | 0.95 | - | - | - | 11.4 | - | 12.0 | - | 16.8 | - | - | 54.8 | ||
SM13 | WGS | - | 25.0 | - | - | - | - | 3.1 | 21.9 | - | 3.1 | 47 | - | chimeric |
Tide | 0.93 | 27.5 | - | - | - | - | 12.0 | 8.1 | - | - | 38.8 | - | ||
SM17 | WGS | - | - | - | - | - | 56.5 | - | - | - | - | 21.7 | 21.7 | chimeric |
Tide | 0.93 | - | - | - | - | 31.7 | - | - | - | - | 31.7 | 28.2 | ||
SM19 | WGS | - | - | - | - | - | - | - | - | - | - | 17.2 | 82.8 | heterozygous |
Tide | 0.98 | - | - | - | - | - | - | - | - | - | - | 97.0 | ||
SM-CTRL | WGS | - | - | - | - | - | - | - | - | - | - | - | 100.0 | wild type |
sgRNAs | Off-Target All | Coding | Non-Coding | Obs. SNP/Indels |
---|---|---|---|---|
7 | 4 | 1 | 3 | 0 |
1 | 5 | 2 | 3 | 0 |
8 | 6 | 0 | 6 | 0 |
6 | 10 | 4 | 6 | 0 |
Total | 25 | 7 | 18 | 0 |
Genotype | Plant Type | SNP | ||||||
---|---|---|---|---|---|---|---|---|
Genot. Specific | Homoz. | Heteroz. | In Exons | (%) | Per Mbp | Per Mbp (avg) | ||
SM6 | edited | 8141 | 3318 | 4823 | 240 | 0.0010% | 9.6 | 8.95 |
SM13 | edited | 9841 | 3554 | 6287 | 348 | 0.0013% | 8.0 | - |
SM17 | edited | 8589 | 3491 | 5098 | 286 | 0.0011% | 9.1 | - |
SM19 | edited | 8587 | 2945 | 5642 | 249 | 0.0011% | 9.1 | - |
CTRL | in vitro | 7960 | 2969 | 4991 | 236 | 0.0010% | 9.8 | 9.04 |
WT-1 | from seed | 8784 | 2925 | 5859 | 269 | 0.0011% | 8.9 | - |
WT-2 | from seed | 9322 | 3236 | 6086 | 279 | 0.0012% | 8.4 | - |
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Li, R.; Maioli, A.; Yan, Z.; Bai, Y.; Valentino, D.; Milani, A.M.; Pompili, V.; Comino, C.; Lanteri, S.; Moglia, A.; et al. CRISPR/Cas9-Based Knock-Out of the PMR4 Gene Reduces Susceptibility to Late Blight in Two Tomato Cultivars. Int. J. Mol. Sci. 2022, 23, 14542. https://doi.org/10.3390/ijms232314542
Li R, Maioli A, Yan Z, Bai Y, Valentino D, Milani AM, Pompili V, Comino C, Lanteri S, Moglia A, et al. CRISPR/Cas9-Based Knock-Out of the PMR4 Gene Reduces Susceptibility to Late Blight in Two Tomato Cultivars. International Journal of Molecular Sciences. 2022; 23(23):14542. https://doi.org/10.3390/ijms232314542
Chicago/Turabian StyleLi, Ruiling, Alex Maioli, Zhe Yan, Yuling Bai, Danila Valentino, Anna Maria Milani, Valerio Pompili, Cinzia Comino, Sergio Lanteri, Andrea Moglia, and et al. 2022. "CRISPR/Cas9-Based Knock-Out of the PMR4 Gene Reduces Susceptibility to Late Blight in Two Tomato Cultivars" International Journal of Molecular Sciences 23, no. 23: 14542. https://doi.org/10.3390/ijms232314542