Plant materials and growth conditions
Four varieties of Solanum lycopersicum L., i.e., cv. Ailsa Craig (AC), Moneymaker (MM), Super Roma (SR), and Rio Grande (RG), were purchased from Thompson & Morgan (UK: abbreviations used in vector and plant line names). Tomato calli and shoots were grown in a growth chamber under conditions of 21–25◦C with 16 h light at 4000– 8000 lx/8 h dark. Tomato plants were grown in a growth chamber or greenhouse under conditions of 21–25◦C with 16 h light at 7000–10,000 lx/8 h dark.
gRNA and CRISPR/Cas9 vectors
The selected SlIAA9 target sequence in genomic DNA [Solyc04g076850, 5’-GAGCTCAGGCTCGGTCTACC-3’—named gRNA2 in our previous study [19]—was completely identical in all four tomato cultivars. The CRISPR/Cas9 expression vectors pEgP237-2A-GFP or pEgPubi4_237-2A-GFP [19] (Additional file 1: Figure S1A) were used, which comprise a gRNA under control of the Arabidopsis U6 snRNA-26 (AtU6-26) promoter, and an Arabidopsis codon-optimized spCas9 (AtCas9) fused to GFP via a 2A peptide. Cas9 expression is driven by the 2 × CaMV35S promoter with the omega translational enhancer in pEgP237-2A-GFP, and a parsley ubi4-2 promoter was used in pEgPubi4_237-2A-GFP. Cas9 contains the 3 × NLS on its C-terminal. In both vectors, NPT II was used as a selection marker in plants.
Transformation
The CRISPR/Cas9 expression vector pEgP237-2A-GFP or pEgPubi4_237-2A-GFP (Ueta et al., 2017) was introduced into Agrobacterium tumefaciens (GV2260) and used to transform tomato leaf disks as described previously [19, 27]. Briefly, leaf discs prepared from cotyledons were soaked in Agrobacterium infection medium [3% sucrose, 1 × MS (pH 5.7), 0.0003% 2-mercaptoethanol, 100 μM acetosyringone, Agrobacterium (OD600 = 0.01)] for transformation and then cultured on co-culture medium [3% sucrose, 1 × MS (pH 5.7), 40 μM acetosyringone] for several days in the dark. The leaf discs were then transferred to callus induction medium (CIM) 1 [3% sucrose, 1 x MS, 100 mg/L kanamycin, 1.5 mg/L trans-zeatin, and 25 mg/L meropenem] and cultured for 4 weeks for cultivars Ailsa Craig, Moneymaker, and Super Roma. Leaf discs from Rio Grande were incubated in CIM2 [3% sucrose, 1 x MS, 100 mg/L kanamycin, 1 mg/L BA, 0.1 mg/L NAA, and 25 mg/L meropenem] for 2 weeks after co-culture, then transferred to CIM1 for 2 weeks. After the appearance of calli, shoot induction medium [3% sucrose, 1 x MS, 100 mg/L kanamycin, 1.0 mg/L trans-zeatin, and 25 mg/L meropenem] was used for further incubation. Transgenic shoots were transferred to root induction medium [1.5% sucrose, 0.5 × MS, 50 mg/L kanamycin, and 25 mg/L meropenem] for 2-4 weeks, and then to soil pots.
Mutation Analyses in CRISPR/Cas9 Target Sites
Genomic DNA was isolated from tomato leaves and fruits using an SDS-based DNA extraction method. Fragments, including the target sequence, were amplified PCR using PrimeSTAR GXL DNA Polymerase (TaKaRa, Japan). The recognition sequence of AccI is included at the predicted DSB position on the SlIAA9 target sequence. In PCR-RFLP, PCR fragments were digested with AccI (NEB, Japan) and analyzed by agarose-gel electrophoresis. For Sanger sequencing analysis, PCR fragments purified from agarose-gel were cloned by the Seamless ligation cloning extract (SLiCE) method [28] into cloning vector pNEB193 (NEB, Japan). All primers used for PCR are listed in Additional file 1: Table S1.
PCR for detection of null-segregant plants
Nine regions of the T-DNA in the CRISPR/Cas9 vector were selected and amplified using PrimeSTAR GXL polymerase (TaKaRa, Japan). Between 20 and 50 plants in the T1 generation and 8 plants from T2 generations were randomly selected for PCR-based detection of T-DNA regions. All primers used for PCR are listed in Additional file 1: Table S1. The amplicon sizes are also indicated in Additional file 1: Table S1. Technical replicates were performed three times for each line.
Southern blot analysis
Total DNA was extracted from leaves of WT and sliaa9-crispr mutants by a CTAB-based DNA isolation method. Briefly, tomato leaves were crushed after freezing with liquid nitrogen, and incubated in 2% CTAB solution. The lysates were extracted with chloroform: isoamyl alcohol (CI) (24:1), and then 1% CTAB solution was added to the water layer. Total DNAs were obtained by CsCl-EtOH precipitation.
Total DNA was digested with HpaI, which has a single restriction recognition site in the vector (Additional file 1: Figure S1), fractionated on 1% agarose gels, and transferred to nylon membrane (Zeta-Probe, Bio-Rad, USA) by capillary transfer. After UV cross-linking (UVP CL-1000 crosslinker, Analytik Jena, Upland, CA, USA), the membranes were hybridized with DIG-labelled probes for gRNA, Cas9, and LHCB (Solyc02g070970) genes. Probes were amplified using pEgP237-2A-GFP (gRNA and Cas9) or tomato genomic DNA (LHCB) using the primers listed in Additional file 1: Table S1, and labeled with DIG by using a DIG High Prime DNA Labeling and Detection Kit (Roche, Basel, Switzerland). Probe labeling, membrane hybridization, and detection were carried out according to the manufacturer’s instructions. Hybridization signals were detected and imaged with PXi (SYNGENE, Bangalore, India).
Off-target analysis by deep sequencing using MiSeq
Deep sequencing was performed using MiSeq Reagent Kit v2 Nano (Illumina, Japan). Genomic DNAs, including the region of the CRISPR/Cas9 target sites and off-target sequences, were amplified by PCR. One off-target candidate sequence (SL2.50 ch6 :26946923-26946946) was selected by Cas-OT to examine the mutation. PCR products were first separated by electrophoresis, then purified from the agarose gel using the Wizard SV Gel and PCR Clean-Up System (Promega, Japan) and used as templates for a second round of PCR. Second PCR primers were subjected to TruSeq (Illumina, Japan). All primers used for PCR are listed in Additional file 1: Table S1. MiSeq data was analyzed using CLC Genomics Workbench software version 7.5.1 (CLC bio, Japan), mapped on the off-target candidate using Integrative Genomics Viewer (IGV) (Broad Institute).