CRISPR/Cas9-mediated fine-tuning of miRNA expression in tetraploid potato

Abstract

MicroRNAs (miRNAs) are small noncoding RNAs, which modulate the abundance and spatiotemporal accumulation of target mRNAs at transcriptional and post-transcriptional levels and through that play important roles in several biological processes in plants. Here we show that in polyploid species, CRISPR/Cas9 system can be used for fine-tuning of miRNA expression, which can have broader range of applications compared to knock-out mutants. We established the complete pipeline for CRISPR-Cas9-mediated modulation of miRNA expression in potato. It consists of (1) design and assembly of dual sgRNA CRISPR/Cas9 constructs, (2) transient transfection of protoplasts following fast and efficient screening by high resolution melting analysis to select functional sgRNAs, and (3) stable transformation of potato explants with functional sgRNAs and selection of regenerated transgenic lines with desired mutations and desired miRNA abundance based on sequencing and RT-qPCR. We show that miRNA-editing using dual sgRNA approach results in different types of mutations among transgenic lines but also in different alleles of the same plant, which are target site-dependent. The most frequent were short deletions, but we also detected 1-nt insertions (T or G), deletions between two sgRNAs and larger deletions. miRNA abundance correlates with the frequency and type of introduced mutations, as more extensive mutations in more alleles result in lower miRNA abundance. Interestingly, some mutated loci can generate alternative miRNAs, now novel targets were however predicted for those. In all transgenic lines with Cas9 expression, we detected mutations, suggesting high efficiency of Cas9-editing. We confirmed the miRNA-editing efficiency of our optimised approach in two different potato genotypes and three different loci.

References

[1] Allen E, Xie Z, Gustafson AM et al. microRNA-directed phasing during trans-acting siRNA biogenesis in plants. Cell. 2005; 121: 207-21.

[2] Andersson M, Turesson H, Nicolia A et al. Efficient targeted multiallelic mutagenesis in tetraploid potato (Solanum tuberosum) by transient CRISPR-Cas9 expression in protoplasts . Plant Cell Rep. 2017; 36: 117-28.

[3] Andersson M, Turesson H, Olsson N et al. Genome editing in potato via CRISPR-Cas9 ribonucleoprotein delivery. Physiol Plant. 2018; 164: 378-84.

[4] Aquino-Jarquin G. Emerging role of CRISPR/Cas9 technology for MicroRNAs editing in cancer research. Cancer Res. 2017; 77: 6812-7.

[5] Axtell MJ, Meyers BC . Revisiting criteria for plant microRNA annotation in the era of big data. Plant Cell. 2018; 30: 272-84.

[6] Baebler Š, Svalina M, Petek M et al. QuantGenius: implementation of a decision support system for qPCR-based gene quantification. BMC Bioinformatics. 2017; 18: 276.

[7] Basak J, Nithin C . Targeting non-coding RNAs in plants with the CRISPR-Cas technology is a challenge yet worth accepting. Front Plant Sci. 2015; 6: 1-8.

[8] Basso MF, Ferreira PCG, Kobayashi AK et al. MicroRNAs and new biotechnological tools for its modulation and improving stress tolerance in plants. Plant Biotechnol J. 2019; 17: 1482-500.

[9] Bologna NG, Mateos JL, Bresso EG et al. A loop-to-base processing mechanism underlies the biogenesis of plant microRNAs miR319 and miR159. EMBO J. 2009; 28: 3646-56.

[10] Butler NM, Atkins PA, Voytas DF et al. Generation and inheritance of targeted mutations in potato (Solanum tuberosum L.) using the CRISPR/Cas system . PLoS One. 2015; 10: 1-12.

[11] Chauvin L, Sevestre F, Lukan T et al. Gene Editing in Potato Using CRISPR-Cas9 Technology. In: Dobnik D, Gruden K, Ramšak Ž, Coll A, eds. Solanum Tuberosum: Methods and Protocols vol 2354. Springer US: New York, NY, 2021, 331-51.

[12] Chung PJ, Chung H, Oh N et al. Efficiency of recombinant CRISPR/rCas9-mediated miRNA gene editing in Rice. Int J Mol Sci. 2020; 21: 1-14.

[13] Cuperus JT, Montgomery TA, Fahlgren N et al. Identification of MIR390a precursor processing-defective mutants in Arabidopsis by direct genome sequencing. Proc Natl Acad Sci U S A. 2010; 107: 466-71.

[14] Dai X, Zhuang Z, Zhao PX . PsRNATarget: a plant small RNA target analysis server (2017 release). Nucleic Acids Res. 2018; 46: W49-54.

[15] Damodharan S, Corem S, Gupta SK et al. Tuning of SlARF10A dosage by sly-miR160a is critical for auxin-mediated compound leaf and flower development. Plant J. 2018; 96: 855-68.

[16] Danilo B, Perrot L, Mara K et al. Efficient and transgene-free gene targeting using agrobacterium-mediated delivery of the CRISPR/Cas9 system in tomato. Plant Cell Rep. 2019; 38: 459-62.

[17] De Oliveira LFV, Christoff AP, Margis R . isomiRID: a framework to identify microRNA isoforms. Bioinformatics. 2013; 29: 2521-3.

[18] Deng F, Zeng F, Shen Q et al. Molecular evolution and functional modification of plant miRNAs with CRISPR. In: Trends in Plant Science.In Press, 2022, Corrected Proof. Elsevier.

[19] FAO Statistical Database. 2021. Available online: http://www.fao.org/faostat/en/#home

[20] Fauser F, Schiml S, Puchta H . Both CRISPR/Cas-based nucleases and nickases can be used efficiently for genome engineering in Arabidopsis thaliana . Plant J. 2014; 79: 348-59.

[21] Fossi M, Comai L . Widespread genome instability in Solanum tuberosum plants regenerated from protoplasts. bioRxiv. 2018; 1-14.

[22] González MN, Massa GA, Andersson M et al. Comparative potato genome editing: agrobacterium tumefaciens-mediated transformation and protoplasts transfection delivery of CRISPR/Cas9 components directed to StPPO2 gene. Plant Cell Tissue Organ Cult. 2021; 145: 291-305.

[23] Hsiau T, Maures T, Waite K et al. Inference of CRISPR edits from sanger trace data. bioRxiv. 2018; 1-14.

[24] Jacobs TB, LaFayette PR, Schmitz RJ et al. Targeted genome modifications in soybean with CRISPR/Cas9. Biotechnology. 2015; 15: 1-10.

[25] Jodder J . Regulation of pri-MIRNA processing: mechanistic insights into the miRNA homeostasis in plant. Plant Cell Rep. 2021; 40: 783-98.

[26] Kozomara A, Birgaoanu M, Griffiths-Jones S . MiRBase: from microRNA sequences to function. Nucleic Acids Res. 2019; 47: D155-62.

[27] Križnik M, Petek M, Dobnik D et al. Salicylic acid perturbs sRNA-gibberellin regulatory network in immune response of potato to potato virus Y infection. Front Plant Sci. 2017; 8: 1-14.

[28] Li J, Li H, Chen J et al. Toward precision genome editing in crop plants. Mol Plant. 2020; 13: 811-3.

[29] Li S, Castillo-González C, Yu B et al. The functions of plant small RNAs in development and in stress responses. Plant J. 2017; 90: 654-70.

[30] Liu Q, Wang F, Axtell MJ . Analysis of complementarity requirements for plant MicroRNA targeting using a Nicotiana benthamiana quantitative transient assay. Plant Cell. 2014; 26: 741-53.

[31] Lorenz R, Bernhart SH, Siederdissen CH et al. ViennaRNA package 2.0. Algorithms for Molecular Biology. 2011; 6: 1-14.

[32] Lu G, Moriyama EN . Vector NTI, a balanced all-in-one sequence analysis suite. Bioinformatics. 2004; 5: 378-88.

[33] Lukan T, Baebler Š, Maruša P-N et al. Cell death is not sufficient for the restriction of potato virus Y spread in hypersensitive response-conferred resistance in potato. Front Plant Sci. 2018a; 9: 168.

[34] Lukan T, Machens F, Coll A et al. Plant X-tender: an extension of the AssemblX system for the assembly and expression of multigene constructs in plants. PLoS One. 2018b; 13: e0190526.

[35] Lukan T, Pompe-Novak M, Baebler Š et al. Precision transcriptomics of viral foci reveals the spatial regulation of immune-signaling genes and identifies RBOHD as an important player in the incompatible interaction between potato virus Y and potato. Plant J. 2020; 104: 645-61.

[36] Lukan T, Veillet F, Križnik M et al. Sequences and secondary structures of mutated pre-miRNAs from CRISPR/Cas9-edited MIR160a, MIR160b and MIR390a genes of potato [data set]. Zenodo. 2022.

[37] Lukan T, Županič A, Mahkovec Povalej T et al. Chloroplast redox state changes indicate cell-to-cell signalling during the hypersensitive response. bioRxiv. 2021.

[38] Molla KA, Sretenovic S, Bansal KC et al. Precise plant genome editing using base editors and prime editors. Nature Plants. 2021; 7: 1166-87.

[39] Nadakuduti SS, Buell CR, Voytas DF et al. Genome editing for crop improvement - applications in clonally propagated polyploids with a focus on potato (Solanum tuberosum L.) . Front Plant Sci. 2018; 9: 1-11.

[40] Natarajan B, Kalsi HS, Godbole P et al. MiRNA160 is associated with local defense and systemic acquired resistance against Phytophthora infestans infection in potato. J Exp Bot. 2018; 69: 2023-36.

[41] Neilsen CT, Goodall GJ, Bracken CP . IsomiRs - the overlooked repertoire in the dynamic microRNAome. Trends Genet. 2012; 28: 544-9.

[42] Nicolia A, Fält AS, Hofvander P et al. Protoplast-Based Method for Genome Editing in Tetraploid Potato. In: Tripodi P, ed. Crop Breeding. Methods in Molecular Biology vol 2264. Humana: New York, NY, 2021.

[43] Pan C, Ye L, Qin L et al. CRISPR/Cas9-mediated efficient and heritable targeted mutagenesis in tomato plants in the first and later generations. Sci Rep. 2016; 6: 1-10.

[44] Pauwels L, De Clercq R, Goossens J et al. A dual sgRNA approach for functional genomics in Arabidopsis thaliana . G3: Genes, Genomes, Genetics. 2018; 8: 2603-15.

[45] Peng A, Chen S, Lei T et al. Engineering canker-resistant plants through CRISPR/Cas9-targeted editing of the susceptibility gene CsLOB1 promoter in citrus. Plant Biotechnol J. 2017; 15: 1509-19.

[46] Petek M, Rotter A, Kogovšek P et al. Potato virus Y infection hinders potato defence response and renders plants more vulnerable to Colorado potato beetle attack. Mol Ecol. 2014; 23: 5378-91.

[47] Rojas AML, Drusin SI, Chorostecki U et al. Identification of key sequence features required for microRNA biogenesis in plants. Nat Commun. 2020; 11: 1-11.

[48] Schaart JG, van de Wiel CCM, Smulders MJM . Genome editing of polyploid crops: prospects, achievements and bottlenecks. Transgenic Res. 2021; 30: 337-51.

[49] Song L, Axtell MJ, Fedoroff NV . RNA secondary structural determinants of miRNA precursor processing in Arabidopsis. Curr Biol. 2010; 20: 37-41.

[50] Stocks MB, Mohorianu I, Beckers M et al. The UEA sRNA workbench (version 4.4): a comprehensive suite of tools for analyzing miRNAs and sRNAs. Bioinformatics. 2018; 34: 3382-4.

[51] Tadege M, Lin H, Bedair M et al. STENOFOLIA regulates blade outgrowth and leaf vascular patterning in Medicago truncatula and Nicotiana sylvestris . Plant Cell. 2011; 23: 2125-42.

[52] Veillet F, Chauvin L, Kermarrec MP et al. The Solanum tuberosum GBSSI gene: a target for assessing gene and base editing in tetraploid potato. Plant Cell Rep. 2019a; 38: 1065-80.

[53] Veillet F, Durand M, Kroj T et al. Precision breeding made real with CRISPR: illustration through genetic resistance to pathogens. Plant Communications. 2020a; 1: 100102.

[54] Veillet F, Kermarrec MP, Chauvin L et al. CRISPR-induced indels and base editing using the Staphylococcus aureus Cas9 in potato. PLoS One. 2020b; 15: 1-11.

[55] Veillet F, Perrot L, Chauvin L et al. Transgene-free genome editing in tomato and potato plants using agrobacterium-mediated delivery of a CRISPR/Cas9 cytidine base editor. Int J Mol Sci. 2019b; 20: 1-10.

[56] Veillet F, Perrot L, Guyon-Debast A et al. Expanding the CRISPR toolbox in P. patens using SpCas9-NG variant and application for gene and base editing in Solanaceae crops. Int J Mol Sci. 2020c; 21: 1-9.

[57] Wang S, Zhang S, Wang W et al. Efficient targeted mutagenesis in potato by the CRISPR/Cas9 system. Plant Cell Rep. 2015; 34: 1473-6.

[58] Wu L, Zhou H, Zhang Q et al. DNA methylation mediated by a MicroRNA pathway. Mol Cell. 2010; 38: 465-75.

[59] Xia R, Xu J, Meyers BC . The emergence, evolution, and diversification of the miR390-TAS3-ARF Pathway in land plants. Plant Cell. 2017; 29: 1232-47.

[60] Zebell SG . Excising the mystery of single-guide RNA processing. Plant Physiol. 2020; 184: 572-3.

[61] Zhang BH, Pan XP, Cox SB et al. Evidence that miRNAs are different from other RNAs. Cell Mol Life Sci. 2006; 63: 246-54.

[62] Zhang D, Zhang Z, Unver T et al. CRISPR/Cas: a powerful tool for gene function study and crop improvement. J Adv Res. 2021; 29: 207-21.

[63] Zhang W, Gao S, Zhou X et al. Multiple distinct small RNAs originate from the same microRNA precursors. GBC. 2010; 11: R81.

[64] Zhou X, Zha M, Huang J et al. StMYB44 negatively regulates phosphate transport by suppressing expression of PHOSPHATE1 in potato. J Exp Bot. 2017; 68: 1265-81.

[65] Zhu H, Zhou Y, Castillo-gonzález C et al. Bi-directional processing of pri-miRNAs with branched terminal loops by Arabidopsis dicer-like1. Nat Struct Mol Biol. 2014; 20: 1106-15.