Plant Materials and Growth Conditions
Nicotiana tabacum L. variety ‘Hong Hua Da Jin Yuan’ (hereinafter referred to as HD) plants were grown at 50% − 60% relative humidity and 25°C 16 h/8 h light/dark cycle in a chamber for 3 weeks before leaves were used for Agrobacterium transformation .
All the primers used for in this study were provided in Supplementary Table S1. Agrobacterium tumefaciens strain LBA4404 harboring the binary vector pORE-Cas9, which contains the CRISPR/Cas9 system, was kindly provided by Prof. Qingyou Xia (Southwestern University, Chongqing, China).
To inspect the efficacy of the OsU3-tRNA, AtU6-tRNA and AtU6 promoters in driving the expression of sgRNA, the OsU3-tRNA-sgRNA-TpolyT, and AtU6-tRNA-sgRNA-TpolyT sequences were synthesized by Nanjing GenScript Co (GenScript, Nanjing), and cloned into the pORE-Cas9 vector instead of the AtU6-sgRNA-TpolyT via the HindIII and SbfI sites through homologous recombination (HR) using the In-Fusion HD cloning kit (Takara), getting the two new vectors OsU3-tRNA and AtU6-tRNA, respectively. Then the tobacco PDS sgRNA was inserted into the BsaI site of the 3 vectors by restriction digestion and ligation method, obtaining the vectors of OsU3-tRNA-/AtU6-tRNA-/AtU6-PDS, respectively (Fig. 1a). The correct clones were confirmed by sequencing with primer RB-F. Similarly, to further optimize the vector, the tobacco NtLHT1 was ligated at the BsaI site in the OsU3-tRNA vector and the AtUb10-Ros1-TNOS expression cassette was synthesized (GenScript) and fused into OsU3-tRNA-LHT1 vector at NotI and KpnI sites by HR. The improved vector was named pOREU3TR-NtLHT1 and the correct clones were identified with EXT-F primer (Fig. 2).
The CRISPR vectors were introduced into the Agrobacterium strain LBA4404, and Agrobacterium-mediated transformation of HD leaves was performed by following the protocol of leaf disc transformation [31, 32]. The tobacco calluses were subcultured on the MS3 medium plates (4.4 g l− 1 MS medium including vitamins, 30 g l− 1 sucrose, 2 mg l− 1 benzylaminopurine, 0.5mg l− 1 Naphthaleneacetic acid, 250 mg l− 1 carbenicillin, 50 mg l− 1 kanamycin and 4 g l− 1 phytagel, pH 5.8) every 2 weeks until the development of kanamycin-resistant calluses. The resistant calluses were sampled for gDNA isolation, and Cas9-1F/R, PDS-1F/R primers (Supplementary Table S1) were used to identify T-DNA insertions and the editing forms of target fragments by PCR amplification and Sanger sequencing. After culture for 6–8 weeks, the induced regenerated shoots were excised and transferred to the rooting medium (4.4 g l− 1 MS medium including vitamins, 30 g l− 1 sucrose, 250 mg l− 1 carbenicillin, 100 mg l− 1 kanamycin and 4 g l− 1 phytagel, pH 5.8), and grown for 2–3 weeks to a height of 5–6 cm before transfer to soil. The regenerated plants were sampled for gDNA extraction and molecular detection. For the pOREU3TR-NtLHT1 vector, the T0 plants were visually screened for color phenotype at calli culture stage and at different growing stages. Seeds from each individual T0 plants containing dark red color in stems and leaves were harvested separately. Cas9-1F/R and LHT1-1F/R primers (Supplementary Table S1) were used for the detection of transgenes and editing forms.
Total RNA of HD and T0 plants of OsU3-tRNA-/AtU6-tRNA-/AtU6-PDS were extracted using Eastep Super Total RNA Isolation Kit (Promega, Shanghai, China), and cDNA was synthesized using the HiScript II Q RT SuperMix for qPCR Kit (Vazyme) following the manufacturer’s protocols. qRT-PCR assays were performed to determine the expression levels of the sgRNA in tobacco leaves, and tobacco NtEF1 (Ntab0421890) was used as internal control. The Primers used for qRT-PCR were described in Supplementary Table S1. The relative expression levels were calculated using the 2−ΔΔCTmethod.
Detection Of T Plants
We randomly selected the T1 progenies of 3 homozygous mutant T0 plants of pOREU3TR-NtLHT1 to determine the efficiency of the new CRISPR/Cas9 system in editing the target genes. Cas9-1F/R was used to detect the presence of the T-DNA, and LHT1-1F/R was used to amplify part of the NtLHT1 gene from the normal green T1 plants. We also obtained the seeds separately from the transgene-free and gene-edited T1 pants.
Characterization Of T Plants
The wild type HD and T2 plants of pOREU3TR-NtLHT1 were planted in soil in the greenhouse. We directly measured the plant height of the plants at rosette stage, flower-bud appearing stage and mature stage, and the tobacco leaves of these stages were sampled to determine the amino acid contents by HPLC (Agilent 1100, USA) .