A Simple and Efficient Protocol to Transform and Regenerate CRISPR-Cas9-Mediated Genome-Edited Tomato Plants

Chapter 14 A Simple and Efficient Protocol to Transform and Regenerate CRISPR-Cas9-Mediated Genome-Edited Tomato Plants Eduardo Martı ´nez-Estrada, Daniela de la Mora-Franco, and Stefan de Folter Abstract CRISPR-Cas9-mediated genome editing has revolutionized functional genomics and crop improvement. However, to maximize the adoption of the CRISPR-Cas9 technology, an efficient method to transfor m and regenerate genetically edited plants is necessary. In this protocol, we describe a detailed method to generate a CRISPR-Cas9 construct based on the Golden Gate cloning system and a simple and efficient method to transform and regenerate tomato plants from cotyledons co-cultured with Agrobacterium. Our protocol allows the production of at least ten Cas-positive independent lines from one hundred cotyledons. This protocol is routinely used in our laboratory to obtain tomato mutant lines and has been proven effective across several genotypes. Key words Solanum lycopersicum, Agrobacterium transformation, Golden Gate cloning, CRISPRCas9, Functional genomics, Genome editing 1 Introduction With the rapid development of sequencing technologies, the genomes of many important plants have been sequenced. This wealth of information will accelerate studies on plant development and functional genomics, genomes and gene evolution, and domestication processes, among others [1]. Taking advantage of this genomic information, CRISPR-Cas9-mediated genome editing has been widely used in many crop species in recent years, including tomato [2, 3]. In CRISPR-Cas9 technology, the endonuclease Cas9 is directed to the target site by a single guide RNA (sgRNA) and cleaves the DNA double strands, generating a targeted doublestranded break [4]. This triggers inherent DNA repair mechanisms in cells: non-homologous end joining (NHEJ) or homologydirected repair (HDR), being the NHEJ pathway dominant in Stefan de Folter (ed.), Plant MicroRNAs: Methods and Protocols, Methods in Molecular Biology, vol. 2900, https://doi.org/10.1007/978-1-0716-4398-3_14, © The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2025 213 most plant cells [5]. The NHEJ mechanism is error-prone and allows random small insertions or deletions and substitutions that can generate frameshift mutations and abolish protein function if such errors occur in open-reading frames [6, 7]. It is relevant to point out that not only coding sequences can be targeted. Regulatory elements that control gene expression levels and patterns can be targets for mutation, and hence, novel alleles and beneficial quantitative trait variations for crop improvement can be generated [8]. Additionally, using CRISPR-Cas9, it is now feasible to directly generate miRNA knockout mutants [9]. Finally, since sgRNAs and the Cas9 gene can be eliminated in the next T1 generation upon segregation, CRISPR-Cas9 is considered a safe, rapid, and environmentally friendly next-generation breeding technology in crop plants [7]. 214 Eduardo Martı ´nez-Estrada et al. To maximize the adoption of the CRISPR-Cas technique, an efficient protocol to transform and regenerate edited plants is necessary. In this sense, to deliver the CRISPR-Cas9 system into tomato plants, the Agrobacterium-mediated transformation followed by regeneration via in vitro culture is the most used method [10–12]. However, also there exist options like polyethylene glycol (PEG)-based protoplast-mediated transformation and particle bombardment or biolistic methods [7, 13]. In this protocol, we describe a detailed method to generate a classical CRISPR-Cas9 construct and a simple and efficient method to transform and regenerate lines from tomato cotyledons co-cultured with Agrobacterium. The CRISPR-Cas9 construct protocol is based on the Golden Gate cloning system [14]. All the plasmids needed are available in Addgene (https://www.addgene. org/) and enable the production of constructs with one to four sgRNA in a single plasmid. In this protocol an example of the assembly of a construct with two sgRNAs will be used; however, we also describe the plasmids for a construct of one or four sgRNAs. On the other hand, the transformation and regeneration method allows the production of at least ten Cas-positive independent lines from one hundred cotyledons. 2 Materials 2.1 CRISPR-Cas9 System 1. Individual plasmids from the MoClo Toolkit obtained from Addgene (www.addgene.org): a Carbenicillin or ampicillin 100 μg/mL a Carbenicillin or ampicillin 100 μg/mL a Carbenicillin or ampicillin 100 μg/mL a Carbenicillin or ampicillin 100 μg/mL CRISPR-Cas9-Mediated Genome Editing in Tomato 215 Plasmid Addgene catalog number Resistance Description Size (bp) ng for 40 fm Guide template pICH86966 46,966 Kanamycin 50 μg/mL Template used for initial PCR 6552 174 Level 0 components pICSL01009 46,968 Spectinomycin 50 μg/mL Plasmid containing AtU6 2323 61.5 Level 1 position plasmids to clone sgRNA pICH47751 48,002 Plasmid to clone guide, position 1 4968 133 pICH47761 48,003 Plasmid to clone guide, position 2 4968 133 pICH47772 48,004 Plasmid to clone guide, position 3 4968 133 pICH47781 48,005 Plasmid to clone guide, position 4 4968 133 Level 1 components pICH47732 51,144 Carbenicillin or ampicillin 100 μg/mL Nosp::NPT2 OCST 6234 166 pICH47742 49,771 Carbenicillin or ampicillin 100 μg/mL 35Sp::Cas9-NOST 9623 251 Linkers pICH41766 48,018 Spectinomycin 50 μg/mL Linker for 1 guide reaction 3318 87 pICH41780 48,019 Spectinomycin 50 μg/mL Linker for 2 guide reaction 3318 87 pICH41822 48,021 Spectinomycin 50 μg/mL Linker for 4 guide reaction 3318 87 Level 2 binary vector pAGM4723 48,015 Kanamycin 50 μg/mL Binary vector for transformation 12,773 333 a IPTG and X-Gal should be added for blue-white screening 216 Eduardo Martı ´nez-Estrada et al. 2. High-fidelity polymerase and all needed components for PCR (water, buffer, dNTPs, MgCl 2 , etc.). 3. Restriction endonucleases: BsaI, BpiI/ BbsI. 4. T4 DNA ligase with buffer. 5. 1% agarose gels. 6. Competent E. coli (DH5α or others). 7. Competent Agrobacterium (GV3101 or others). 8. LB plates with ampicillin or carbenicillin (100 μg/mL), X-gal (40 μg/mL) and IPTG (100 μm) (see Notes 1 and 2). 9. LB plates with kanamycin (50 μg/mL) (see Note 3). 10. LB plates with kanamycin (50 μg/mL) and rifampicin (50 μg/ mL) (see Note 3). 11. Forward primers for sgRNA synthesis (primer design is described below in the Methods section). 12. Reverse primer for sgRNA synthesis: (a) sgRNA-Rv: 5′-tgtggtctcaagcgtaatgccaactttgtac-3′. 13. Primers to confirm Level 1 construct: (a) U6-Fw: 5′-ggagtgatcaaaagtcccac-3′. (b) sgRNA-Rv: 5′-tgtggtctcaagcgtaatgccaactttgtac-3′ (same primer as in point 12). 14. Primers to confirm Cas9 in Level 2 construct: (a) Cas9-Fw: 5′-ctgacgtaagggatgacgcac-3′. (b) Cas9-Rv: 5′-catctcattactaaagatctcc-3′. 15. Primers to confirm sgRNAs in Level 2 construct: (a) U6-Fw: 5′-ggagtgatcaaaagtcccac-3′ (same primer as in point 13a). (b) Backbone-Rv: 5′-cggataaaccttttcacgcc-3′. 16. Primer for sequencing the final Level 2 construct: (a) Backbone-Rv: 5′-cggataaaccttttcacgcc-3′ (same primer as in point 15b). 2.2 Tomato Transformati on and Regeneration 2.2.1 Medium for Agrobacterium Tumefaciens Culture 1. Luria-Bertani (LB) medium. 2. Yeast Extract Peptone (YEP) liquid medium. 3. Selection agent. CRISPR-Cas9-Mediated Genome Editing in Tomato 217 2.2.2 Plant Material 1. Seeds of Solanum lycopersicum for transformation. This protocol has been evaluated in Micro-Tom (MT), M82, and Moneyberg cultivars. 2.2.3 In-Vitro Culture 1. Sterilization solution: 20% commercial bleach solution (v/v) in sterile distilled water with a drop (0.01%) of Tween 20. 2. Sterile distilled water. 3. Germination medium: 50% Murashige and Skoog (MS) medium, 15 g/L sucrose. Adjust the medium pH to 5.8 and add 7 g/L of agar before sterilizing (see Notes 4 and 5). 4. Co-culture medium: 100% MS medium, 30 g/L sucrose. Adjust the medium pH to 5.8 and add 7 g/L of agar before sterilizing. After sterilizing and cooling the medium to below 55 °C, add 0.2 mg/L of 2,4-Dichlorophenoxyacetic acid (2,4-D) and 0.1 mg/L of kinetin (see Note 6). 5. Selection and regeneration medium: 100% MS medium, 30 g/ L sucrose. Adjust the medium pH to 5.8 and add 7 g/L of agar before sterilizing. After sterilizing and cooling the medium to below 55 °C, add 2 mg/L of trans-Zeatin, 25 mg/L of meropenem and the appropriate selection agent (75 mg/L of kanamycin for the pAGM4723 binary vector) (see Note 7). 6. Selective rooting medium: 100% MS medium, 30 g/L sucrose. Adjust the medium pH to 5.8 and add 7 g/L of agar before sterilizing. After sterilizing and cooling the medium to below 55 °C, add 1 mg/L of indol-acetic acid (IAA), 30 mg/L of meropenem and the appropriate selection agent (see Note 8). 7. Sterile paper towels cut in half and filter paper (or paper sheets cut into squares) (see Note 9). 8. Glass jars of 10 cm in height or magenta boxes. 9. Petri dishes of 150 and 100 mm. 2.3 Acclimatization and Greenhouse 1. Seed trays with lids. 2. Soil mix: Peat-based substrate and perlite (1:1). 3. Plastic pots: 15 cm (or 6 inch) or 21 cm (or 8 inch) in diameter. 4. Commercial plant fertilizer (e.g., Miracle-Gro®) and rooting stimulator (e.g., Raizone plus®). 2.4 Genotyping of Editing Tomato Plantlets 1. CTAB extraction buffer: 100 mM Tris, pH 7.5, 700 mM NaCl, 50 mM EDTA, pH 8.0, 2% cetyltrimethyl ammonium bromide (CTAB). 2. Taq DNA polymerase and all needed components for PCR (water, buffer, dNTPs, MgCl 2 , etc.). 218 Eduardo Martı ´nez-Estrada et al. 3. Cas9 primers (Same primers as in point 14, Subheading 2.1): (a) Cas9-Fw: 5′-ctgacgtaagggatgacgcac-3′. (b) Cas9-Rv: 5′-catctcattactaaagatctcc-3′. 4. Primers that flank the single guide RNA (sgRNA) target site (primer design is described below in Subheading 3.3). 3 Methods 3.1 CRISPR-Cas9 System 1. For the single guide RNA (sgRNA), select a 20 bp target within the gene or region of interest. The target sequence is a consensus of 5′-GNNNN NNNNN NNNNN NNNNN-3′ followed by an NGG sequence at the 3′ end, which is the specific protospacer adjacent motif (PAM) for the Cas9 in the pICH47742 plasmid. sgRNAs can be selected either on the sense or antisense DNA strand. For purposes of this protocol, the first base at the 5′ end of the sgRNA should be G since it is complementary to the pICSL01009 vector, which contains the AtU6 promoter. However, the G at the 5′ end can be replaced with any other base if necessary, but Level 1 transformation could be less efficient (part of the 4 bp overhang). It is important to select a target that does not contain BsaI and BbsI/BpiI restriction sites. To knock out gene expression, select a target near to the transcriptional start site. To modify expression levels, select a target(s) within the promoter region. Alternatively, to disrupt a gene by inducing a significant deletion, select two or more sgRNAs targeting two adjacent sequences within a locus of interest (see Note 10). 2. Design forward sgRNA primer (s) as follows (primer length is 56 bp and does not include the PAM motif): Forward sgRNA primer: The primer includes a BsaI site (blue) and the 20 bp guide sequence (red). sgRNA sequence does not include the protospacer adjacent motif (PAM). 5´-tgtggtctcaATTGNNNNNNNNNNNNNNNNNNNgttttagagctagaaatagcaag-3´ TheprimerincludesaBsaIsite(blue) and the 20 bp guide sequence (red).sgRNA sequence does not include the protospacer adjacent motif (PAM). . 3. Amplify by PCR the sgRNAs using the pICH86966 vector as a template, the forward primer designed in step 2 and sgRNARv as reverse primer. Primers amplify at 60 °C a product of 164 bp. This step is illustrated in Fig. 1. 4. Purify the PCR product with a purification kit. (a). Mix for guide 1 Mix for guide 2 CRISPR-Cas9-Mediated Genome Editing in Tomato 219 Fig. 1 Scheme illustrating the PCR reaction to generate the sgRNA, using the pICH86966 plasmid as a template, the forward primer containing the target sequence, and the reverse primer to clone the scaffold. (Created with BioRender.com) 5. For the Level 1 vector, set up cut-ligation reactions separately for each sgRNA using BsaI enzyme as follows (for this example, 100 ng/uL for plasmid and 5 ng/uL for the PCR fragments were assumed): Final concentration Volume (μL) Water Water –To 10 μL 10× T4 ligase buffer 10× T4 ligase buffer 1×1 μL BsaI (8 U) BsaI (8 U) 8 U 0.8 μL T4 ligase (8 U) T4 ligase (8 U) 8 U 0.8 μL PCR fragment guide 1 (5 ng) PCR fragment guide 2 (5 ng) 40 fmol 1 μL pICH47751 (100 ng) pICH47761 (100 ng) 40 fmol 1.3 μL pICSL01009 (100 ng) pICSL01009 (100 ng) 40 fmol 0.6 μl (b). 16°C50°C80°C12°C 220 Eduardo Martı ´nez-Estrada et al. 37 °C 3:00 4:00 5:00 5:00 Hold 20×1×1× 6. Transform each ligation into E. coli and select white colonies on LB plates with X-Gal, IPTG and Carbenicillin (Fig. 2). Perform PCR using the U6-Fw and sgRNA-Rv to confirm the Level 1 construct. Primers amplify at 60 °C, a product of 239 bp. The pICH47751-guide 1 and pICH47761-guide 2 final length is 4588 bp. Fig. 2 Scheme illustrating the assembling of Level 1 construct for two differen t sgRNas. sgRNAs are placed under the control of Arabidopsis U6 promoter. Negative colonies grow blue; select white colonies. (Created with BioRender.com) (a). (b). 16°C50°C80°C12°C CRISPR-Cas9-Mediated Genome Editing in Tomato 221 7. For the Level 2 vector set up a cut-ligation reaction using BpiI/ BbsI enzyme as follows: Mix for Level 2 reaction Final concentration Volume (μL) Water –To 20 μL 10× T4 ligase buffer 1×2 μL BpiI/BbsI (10 U) 10 U 1 μL T4 ligase (10 U) 10 U 1 μL pICH47732 (100 ng) 40 fmol 1.6 μL pICH47742 (100 ng) 40 fmol 2.5 μL pICH47751-guide 1 (100 ng) 40 fmol 1.2 μL pICH47761-guide 2 (100 ng) 40 fmol 1.2 μL pICH41780 (100 ng) 40 fmol 0.8 μL pAGM4723 (100 ng) 40 fmol 3.3 μL 37 °C 3:00 4:00 5:00 5:00 Hold 20×1×1× 8. Transform the ligation into E. coli and select white colonies on LB plates with kanamycin (Fig. 3). 9. 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