CRISPR/Cas9-mediated multiple gene editing in Brassica oleracea var. capitata using the endogenous tRNA-processing system

Abstract

Cabbage (Brassica oleracea var. capitata) is a biennial plant with strong self-incompatibility and an obligate requirement for prolonged vernalization by exposure to low temperatures to induce flowering. These characteristics significantly increase the difficulty of exploiting novel germplasm induced by physical or chemical mutagens. In this study, we report a CRISPR/Cas9 gene-editing system based on endogenous tRNA processing to induce high efficiency and inheritable mutagenesis in cabbage. Using the phytoene desaturase gene BoPDS, the S-receptor kinase gene BoSRK, and the male-sterility-associated gene BoMS1 as the target genes, multisite and multiple gene mutations were achieved using a construct with tandemly arrayed tRNA-sgRNA architecture to express multiple sgRNAs. The BoSRK3 gene mutation suppressed self-incompatibility completely, converting the self-incompatible line into a self-compatible line. In addition, the BoMS1 gene mutation produced a completely male-sterile mutant, which was highly cross compatible with its nonmutant isoline at the flowering stage as a result of a simultaneous BoSRK3 gene mutation, enabling the economic propagation of the male-sterile line through bee-mediated cross-pollination. Interestingly, higher site mutation efficiency was detected when a guide sequence was inserted into a location in the tandemly arrayed tRNA-sgRNA architecture that was distal from the upstream Pol III promoter. In addition, mutation sites were also detected in the paralogous genes of the BoPDS and BoSRK genes that had fully consistent sequences or base mismatches but beyond the “seed” region in the spacer sequence compared with the target sgRNAs. Collectively, our results demonstrate that the CRISPR/Cas9 system, coupled with an endogenous tRNA-processing system, is an efficient tool to improve cabbage traits.

References

[1] Pacher, M. & Puchta, H. From classical mutagenesis to nuclease-based breeding-directing natural DNA repair for a natural end-product. Plant J. 90, 819-833 (2017).

[2] Xiong, J. S., Ding, J. & Li, Y. Genome-editing technologies and their potential application in horticultural crop breeding. Hortic. Res. 2, 15019 (2015).

[3] Sprink, T., Metje, J. & Hartung, F. Plant genome editing by novel tools: TALEN and other sequence specific nucleases. Curr. Opin. Biotechnol. 32, 47-53 (2015).

[4] Sun, Z. J. et al. Site-specific gene targeting using transcription activator-like effector (TALE)-based nuclease in Brassica oleracea. J. Integr. Plant Biol. 55, 1092-1103 (2013).

[5] Schaeffer, S. M. & Nakata, P. A. The expanding footprint of CRISPR/Cas9 in the plant sciences. Plant Cell Rep. 35, 1451-1468 (2016).

[6] Cong, L. et al. Multiplex genome engineering using CRISPR/Cas systems. Science 339, 819-823 (2013).

[7] Lawrenson, T. et al. Induction of targeted, heritable mutations in barley and Brassica oleracea using RNA-guided Cas9 nuclease. Genome Biol. 16, 258-270 (2015).

[8] Ma, X. L. et al. A robust CRISPR/Cas9 system for convenient, high-efficiency multiplex genome editing in monocot and dicot plants. Mol. Plant. 8, 1274-1284 (2015).

[9] Xie, K. B., Minkenberg, B. & Yang, Y. N. Boosting CRISPR/Cas9 multiplex editing capability with the endogenous tRNA-processing system. PNAS 112, 3570-3575 (2015).

[10] Qi, W. W. et al. High-efficiency CRISPR/Cas9 multiplex gene editing using the glycine tRNA-processing system-based strategy in maize. BMC Biotechnol. 16, 58-65 (2016).

[11] Port, F. & Bullock, S. L. Expansion of the CRISPR toolbox in an animal with tRNA-flanked Cas9 and Cpf1 gRNAs. Preprint at https://www.biorxiv.org/content/early/2016/03/31/046417.1 (2016).

[12] Wang, W., Akhunova, A., Chao, S. M. & Akhunov, E. Optimizing multiplex CRISPR/Cas9-based genome editing for wheat. Preprint at https://www.biorxiv.org/content/early/2016/05/12/051342 (2016).

[13] Wang, P. C. et al. High efficient multi-sites genome editing in allotetraploid cotton (Gossypium hirsutum) using CRISPR/Cas9 system. Plant Biotechnol. J. 16, 137-150 (2017).

[14] Braatz, J. et al. CRISPR-Cas9 targeted mutagenesis leads to simultaneous modification of different homoeologous gene copies in polyploid oilseed rape (Brassica napus). Plant Physiol. 174, 935-942 (2017).

[15] Yang, H., Wu, J. J., Tang, T., Liu, K. D. & Dai, C. CRISPR/Cas9-mediated genome editing efficiently creates specific mutations at multiple loci using one sgRNA in Brassica napus. Sci. Rep. 7, 7489 (2017).

[16] Sun, Q. F. et al. CRISPR/Cas9-Mediated Multiplex Genome Editing of the BnWRKY11 and BnWRKY70 Genes in Brassica napus L.. Int J. Mol. Sci. 19, 2716 (2018).

[17] Liu, S. Y. et al. The Brassica oleracea genome reveals the asymmetrical evolution of polyploid genomes. Nat. Commun. 5, 3930 (2014).

[18] Żur, I. et al. Environmental factors and genotypic variation of self-incompatibility in Brassica oleracea L. var. Capitata. Acta Biol. Crac.-Ser. Bot. 45, 49-52 (2003).

[19] Dhall, R. K. Status of male sterility in vegetables for hybrid development-A Review. Adv. Hort. Sci. 24, 263-279 (2010).

[20] Delorme et al. Characterization of the S locus genes, SLG and SRK, of the Brassica S3 haplotype: identification of a membrane-localized protein encoded by the S locus receptor kinase gene. Plant J. 7, 429-440 (1995).

[21] Chang, F., Wang, Y. X., Wang, S. S. & Ma, H. Molecular control of microsporogenesis in Arabidopsis. Curr. Opin. Plant Biol. 14, 66-73 (2011).

[22] Yang, C. Y., Vizcay-Barrena, G., Conner, K. & Wilson, Z. A. MALE STERILITY1 is required for tapetal development and pollen wall biosynthesis. Plant Cell 19, 3530-3548 (2007).

[23] Li, H. et al. PERSISTENT TAPETAL CELL1 encodes a PHD-finger protein that is required for tapetal cell death and pollen development in rice. Plant Physiol. 156, 615-630 (2011).

[24] Gomez, J. F. & Wilson, Z. A. A barley PHD finger transcription factor that confers male sterility by affecting tapetal development. Plant Biotechnol. J. 12, 765-777 (2014).

[25] Jeong, K., Choi, D. & Lee, J. Fine mapping of the genic male-sterile ms1 gene in Capsicum annuum L.. Theor. Appl. Genet. 131, 183-191 (2018).

[26] Fan, D. et al. Efficient CRISPR/Cas9-mediated targeted mutagenesis in Populus in the first generation. Sci. Rep. 5, 12217 (2015).

[27] Ito, H., Saito, T. & Hatayama, T. Time and temperature factors for the flower formation in cabbage. II. The site of vernalization and the nature of vernalization sensitivity. Tohoku J. Agric. Res. 17, 1-15 (1966).

[28] Gerszberg, A. Tissue culture and genetic transformation of cabbage (Brassica oleracea var. capitata): an overview. Planta. https://doi.org/10.1007/s00425-018-2961-3 (2018).

[29] Liu, X. X. et al. Sequence features associated with the cleavage efficiency of CRISPR/Cas9 system. Sci. Rep. 6, 19675 (2016).

[30] Peterson, B. A. et al. Genome-wide assessment of efficiency and specificity in CRISPR/Cas9 mediated multiple site targeting in Arabidopsis. PLoS ONE 11, e0162169 (2016).

[31] Graham, D. B. & Root, D. E. Resources for the design of CRISPR gene editing experiments. Genome Biol. 16, 260 (2015).

[32] Johnson, R. A., Gurevich, V., Filler, S., Samach, A. & Levy, A. A. Comparative assessments of CRISPR Cas nucleases’ cleavage efficiency in planta. Plant Mol. Biol. 87, 143-156 (2015).

[33] Zhang, Q. et al. Potential high-frequency off-target mutagenesis induced by CRISPR/Cas9 in Arabidopsis and its prevention. Plant Mol. Biol. 96, 445-456 (2018).

[34] Sekine, R., Kawata, T. & Muramoto, T. CRISPR/Cas9 mediated targeting of multiple genes in Dictyostelium. Sci. Rep. 8, 8471 (2018).

[35] Zhang, Q. et al. High-frequency off-target mutagenesis induced by CRISPR/Cas9 in Arabidopsis and its prevention by improving specificity of the tools. https://www.biorxiv.org/content/early/2017/11/27/203489 (2017).

[36] Feng, Z. Y. et al. Multigeneration analysis reveals the inheritance, specificity, and patterns of CRISPR/Cas induced gene modifications in Arabidopsis. PNAS 111, 4632-4637 (2014).

[37] Hsu, P. D. et al. DNA targeting specificity of RNA-guided Cas9 nucleases. Nat. Biotechnol. 31, 827-832 (2013).

[38] Nasrallah, J. B., Rundle, S. J. & Nasrallah, M. E. Genetic evidence for the requirement of the Brassica S-locus receptor kinase gene in the self-incompatibility response. Plant J. 5, 373-384 (1994).

[39] Fujimoto, R., Sugimura, T. & Nishio, T. Gene conversion from SLG to SRK resulting in self-compatibility in Brassica rapa. FEBS Lett. 580, 425-430 (2006).

[40] Shu, J. S. et al. Detection of the diversity of cytoplasmic male sterility sources in Broccoli (Brassica oleracea var. italica) using mitochondrial markers. Front Plant Sci. 7, 927 (2016).

[41] Parkash, C. et al. ‘Ogura’-based ‘CMS’ lines with different nuclear backgrounds of cabbage revealed substantial diversity at morphological and molecular levels. 3 Biotech 8, 27 (2018).

[42] Tatum, L. A. The southern corn leaf blight epidemic. Science 171, 1113-1116 (1971).

[43] Perez-Prat, E. & van Lookeren Campagne, M. M. Hybrid seed production and the challenge of propagating male-sterile plants. Trends Plant Sci. 7, 199-203 (2002).

[44] Wu, Y. et al. Development of a novel recessive genetic male sterility system for hybrid seed production in maize and other cross-pollinating crops. Plant Biotechnol. J. 14, 1046-1054 (2016).

[45] Chang, Z. Y. et al. Construction of a male sterility system for hybrid rice breeding and seed production using a nuclear male sterility gene. PNAS 113, 14145-14150 (2016).

[46] Dang, Y. et al. Optimizing sgRNA structure to improve CRISPR-Cas9 knockout efficiency. Genome Biol. 16, 280 (2015).

[47] Bhalla, P. L. & Singh, M. B. Agrobacterium-mediated transformation of Brassica napus and Brassica oleracea. Nat. Protoc. 3, 181-189 (2008).

[48] Rogers, S. O. & Bendich, A. J. Extraction of DNA from milligram amounts of fresh, herbarium and mummified plant tissues. Plant Mol. Biol. 5, 69-76 (1985).

[49] Ma, X. L., Chen, L. T., Zhu, Q. L., Chen, Y. L. & Liu, Y. G. Rapid decoding of sequence-specific nuclease-induced heterozygous and biallelic mutations by direct sequencing of PCR products. Mol. Plant 8, 1285 (2015).

[50] Li, S. F., Iacuone, S. & Parish, R. W. Suppression and restoration of male fertility using a transcription factor. Plant Biotech. J. 5, 297-312 (2007).