Implementing the CRISPR/Cas9 Technology in Eucalyptus Hairy Roots Using Wood-Related Genes
Abstract
:1. Introduction
2. Results
2.1. Genotyping Revealed High Knock-Down Rate in CCR1 but High Knock-out Rate in IAA9A
2.1.1. High Knock-Down Rate for CCR1 Editing
2.1.2. High Knock-Out Rate in IAA9A Lines
2.1.3. Mutation Spectra Vary Among sgRNA Targets
2.2. Phenotyping Revealed Expected Alterations of Lignification in CCR1-Edited Lines
2.2.1. Combination of FTIR Spectroscopy and Multivariate Analyses of CCR1-Edited Hairy Root Lines
2.2.2. Histochemical Characterization of CCR1-Edited Hairy Root Lines
3. Discussion
4. Materials and Methods
4.1. Plant Material
4.2. CRISPR/Cas9 Targeted Mutagenesis System Selection and Pipeline
4.3. CRISPR/Cas9 Target Site Selection and sgRNAs Design
4.4. CRISPR/Cas9 Constructs Assembly
4.5. Agrobacterium Rhizogenes-Mediated Transformation
4.6. DNA Isolation, PCR Amplification and Mutation Identification
4.7. FTIR Analyses
4.8. Histochemical Analysis
Supplementary Materials
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
Abbreviations
BSA | Bovine serum albumin |
CaMV | Cauliflower mosaic virus |
CCR | Cinnamoyl CoA Reductase |
CRISPR | The Clustered Regularly Interspaced Short Palindromic Repeats |
CTAB | Cetyl Trimethylammonium Bromide |
DSDecode | Degenerate Sequence Decoding |
FT-IR | Fourier Transformed Infra-red spectroscopy |
Kana | Kanamycin |
ORF | Open reading frame |
PAM | Protospacer Adjacent Motif |
PLS-DA | Partial Least Square Analysis |
sgRNA | Single guide RNA |
SNP | Single Nucleotide Polymorphism |
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Gene | Edited Plants/Total Transgenic Plants | Plants with All Alleles Altered (no WT) | Plants with one WT Allele | WT/WT | |||
---|---|---|---|---|---|---|---|
Homoz. (A1/A1) | Biallelic (A1/A2) | Chimera (A1/A2/A3...) | Monoallelic (WT/A1) | Chimera (WT/A1/A2...) | |||
CCR1 | 24/24 (100%) | 0 | 0 | 1 (4.2%) | 10 (41.2%) | 13 (54.2%) | 0 |
IAA9A | 12/13 (92.3%) | 0 | 7 (53.8%) | 4 (30.8%) | 1 (7.7%) | 0 | 1 (7.7%) |
Gene | Total Sequenced Subclones | Edited Subclones | Editions in sgRNA1 | Editions in sgRNA2 | Editions in sgRNA1&2 | Large Deletions |
---|---|---|---|---|---|---|
CCR1 | 278 | 89 (32.0%) | 46 (51.7%) | 65 (73.0%) | 22 (24.7%) | 19 (21.3%) |
IAA9A | 95 | 88 (92.6%) | 84 (95.5%) | 79 (89.9%) | 75 (85.2%) | 27 (30.7%) |
Gene | Edited Clones | Presumed Significant Modifications | Presumed Less Significant Modifications (15 bp Indel, Substitution without Shift) | |
---|---|---|---|---|
Reading Frame Shift 1 | ≥15 bp Indel | |||
CCR1 | 89 | 60 (67.4%) | 22 (24.7%) | 27 (30.3%) |
62 (69.7%) | ||||
IAA9A | 88 | 75 (85.2%) | 50 (56.8%) | 12 (13.6%) |
76 (86.4%) |
Gene | Total Edited Clones | Total Edition Types | Deletion | Insertion | Substitution | Expected Large Deletion | |||||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Small Deletion (≤15 bp) | Large Deletion (>15 bp) | Small Insertion (≤15 bp) | Large Insertion (>15 bp) | Small Substitution (≤15 bp) | Large Substitution (>15 bp) | ||||||||||||||||
sg1 | sg2 | sg1&2 | sg1 | sg2 | sg1&2 | sg1 | sg2 | sg1&2 | sg1 | sg2 | sg1&2 | sg1 | sg2 | sg1&2 | sg1 | sg2 | sg1&2 | ||||
CCR1 | 89 | 43 | 12 | 40 | 0 | 19 | 21 | 19 | 3 | 1 | 0 | 0 | 0 | 0 | 24 | 5 | 0 | 0 | 0 | 0 | 19 (21.3%) |
52 (58.4%) | 21 (23.6%) | 4 (4.5%) | 0 | 29 (32.6%) | 0 | ||||||||||||||||
IAA9A | 88 | 20 | 29 | 39 | 19 | 38 | 27 | 27 | 5 | 11 | 0 | 12 | 0 | 0 | 12 | 2 | 0 | 0 | 0 | 0 | 27 (30.7%) |
49 (55.7%) | 38 (43.2%) | 16 (18.2%) | 12 (13.6%) | 14 (15.9%) | 0 |
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Dai, Y.; Hu, G.; Dupas, A.; Medina, L.; Blandels, N.; San Clemente, H.; Ladouce, N.; Badawi, M.; Hernandez-Raquet, G.; Mounet, F.; et al. Implementing the CRISPR/Cas9 Technology in Eucalyptus Hairy Roots Using Wood-Related Genes. Int. J. Mol. Sci. 2020, 21, 3408. https://doi.org/10.3390/ijms21103408
Dai Y, Hu G, Dupas A, Medina L, Blandels N, San Clemente H, Ladouce N, Badawi M, Hernandez-Raquet G, Mounet F, et al. Implementing the CRISPR/Cas9 Technology in Eucalyptus Hairy Roots Using Wood-Related Genes. International Journal of Molecular Sciences. 2020; 21(10):3408. https://doi.org/10.3390/ijms21103408
Chicago/Turabian StyleDai, Ying, Guojian Hu, Annabelle Dupas, Luciano Medina, Nils Blandels, Hélène San Clemente, Nathalie Ladouce, Myriam Badawi, Guillermina Hernandez-Raquet, Fabien Mounet, and et al. 2020. "Implementing the CRISPR/Cas9 Technology in Eucalyptus Hairy Roots Using Wood-Related Genes" International Journal of Molecular Sciences 21, no. 10: 3408. https://doi.org/10.3390/ijms21103408
APA StyleDai, Y., Hu, G., Dupas, A., Medina, L., Blandels, N., San Clemente, H., Ladouce, N., Badawi, M., Hernandez-Raquet, G., Mounet, F., Grima-Pettenati, J., & Cassan-Wang, H. (2020). Implementing the CRISPR/Cas9 Technology in Eucalyptus Hairy Roots Using Wood-Related Genes. International Journal of Molecular Sciences, 21(10), 3408. https://doi.org/10.3390/ijms21103408