Dataset on nicotine-free, nontransgenic tobacco (Nicotiana tabacuml.) edited by CRISPR-Cas9

This dataset in brief is related to the research letter entitled “Nicotine-free, nontransgenic tobacco (Nicotiana tabacuml.) edited by CRISPR-Cas9” [1]. Cured tobacco products with a significantly reduced nicotine content helps people to overcome their nicotine addiction. Here we summarize additional data and method descriptions of the generation process of a nicotine-free, nontransgenic tobacco plant. This included the cloning, transformation and regeneration of transgenic tobacco plants, followed by the analysis of the nicotine content and genomic modifications caused by CRISPR-Cas9 mediated gene editing. Subsequently, nicotine-free plants were screened for loss of T-DNA cassette, i.e. nontransgenity. Finally, a metabolic footprint was recorded by 1H NMR analysis.


Data
The data shows the generation of a nicotine-free and nontransgenic tobacco plant by CRISPR-Cas9 mediated gene editing. After regeneration of plants and testing them for transgenity ( Fig. 1) the nicotine content was analyzed by GC measurements in wild type, T 0 and T 1 generations (Fig. 2). Genomic analysis of T 1 3.1 plant revealed that not all BBL (berberine bridge enzyme-like) loci were knocked out (BBLe) (Fig. 3). Therefore, further generations were grown and analyzed. The nicotine level of T 3 4.11.1.2 plant was reduced to 0.04 mg g À1 DW [1] (Fig. 4). This was additionally confirmed by GC-MS measurements (Fig. 5). The genomic analysis showed knockout of all BBL-loci [1]. The loss of the T-DNA cassette was proven for plant T 3 4.11.1.2 (Fig. 6). Finally, 1 H NMR analysis showed no significant changes in primary metabolism (Figs. 7e9).  Value of the Data First nicotine-free, nontransgenic tobacco plant that enables the production of non-addictive cured tobacco. Nicotine-free smoking products can support people to overcome nicotine addiction. This technology can be transferred to commercially used N. tabacum varieties as well as N. benthamiana to improve the biotechnological production properties. First technology that eliminates the nicotine content while all other metabolites are not affected. with a light intensity of 110 mM m À2 s À1 . For germination, seeds were surface sterilized with a sodium hypochlorite solution (1% active chlorine) and a few drops of Tween 20 for 10 minutes and washed three times with water before they were plated out on Murashige and Skoog Medium (4.4 g L À1 Murashige and Skoog Medium with Gamborg's Vitamins, 30 g L À1 sucrose, pH 5.8).

Plasmid construction
For the delivery of the CRISPR cassette to N. tabacum plants with A. tumefaciens a binary vector system was used [2]. The chosen sgRNA target sequence (GAAATCAGAGTAAGGTGCGG) for the BBL genes was cloned into the vector pChimera according to the author's instructions and the resulting vector was named pChimera-BBL. The gRNA chimera was subsequently cloned into the vector pCas9-TPC according to the author's instructions resulting in the vector pCas9-BBL, which was used for transformation experiments. All cloned vectors were verified by sequencing.
For the verification of the targeted mutagenesis on genomic level, gene sequences of the six BBL genes that include the target site were amplified with specific primers for each gene from genomic DNA of wildtype and transgenic plants. For sequencing, the gene fragments were either cloned into the vector pDionysos [3] by using the Gibson Assembly method or the PCR product was directly sequenced. Used primers are listed in Table 1.

Plant transformation and regeneration
A. tumefaciens GV3101::pMP90 cells were transformed with the plasmid pCas9-BBL as described previously [4]. Transformation of N. tabacum leaves and the followed plant regeneration were done according to an existing protocol with minor changes [5]. Plants were infiltrated with an OD 600 nm of 0.1 and incubated for 3 days at long day conditions. For plant regeneration infiltrated leaves were surface sterilized, cut into pieces and incubated on shooting medium (2.15 g L À1 Murashige and Skoog Medium with Gamborg's Vitamins, 30 g L À1 sucrose, 0.1 mg L À1 indole-3-butyric acid, 0.8 mg L À1  benzylaminopurine, 250 mg L À1 carbenicillin, 6 mg L À1 DL-phosphinothricin (PPT), 8 g L À1 agar, pH 5.2) under long day conditions. Developed shoots were transferred to rooting medium (2.15 g L À1 Murashige and Skoog Medium with Gamborg's Vitamins, 30 g L À1 Sucrose, 0.5 mg L À1 indole-3-butyric acid, 250 mg L À1 carbenicillin, 6 mg L À1 DL-phosphinothricin (PPT), 8 g L À1 agar, pH 5.2) for approximately 10 days.

Test for transgenic plants
Seeds from regenerated plants were collected and seeded out for growing the T 1 generation. To test if these plants were transgenic, 7 days-old plantlets were sprayed with a solution of 100 mg L À1 PPT    Plants in T 2 generation can be tested for the loss of the CRISPR-Cas9 cassette. For this purpose leaves of T 2 generation plants were surface sterilized for 10 minutes with sodium hypochlorite (0.5% active chlorine) and a few drops of Tween 20 followed by three washing steps with water. Leaf discs were cut out with a cork borer and incubated on selection medium (4.4 g L À1 Murashige and Skoog Medium with Gamborg's Vitamins, 30 g L À1 sucrose, 6 mg L À1 PPT; pH 5.8) under standard growing conditions. Additionally, validation of the loss of the CRISPR cassette was done with PCR. Genomic DNA of the plants was isolated and amplification of the CRISPR cassette was done by the use of primers SS43 and SS61.

Plant extracts
For the extraction of alkaloids a modified version of the extraction protocol from Lewis et al. [6] was used. For the extraction 50 mg or 100 mg of ground leaves with 1 mL of a 2 N NaOH to moisten the sample in a glass vessel with a screw-cap. After 15 minutes of incubation time 5 mL of methyl tert-butyl ether (MTBE) containing 0.4 mg ml À1 quinoline used as an internal standard were added to the sample. Samples were incubated for 2.5 h with shaking at 200 rpm. For layer separation the glass vessels were stored without shaking overnight. The MTBE layer was used for gas chromatographic analysis.

Gas chromatographic analysis
Measurements of plant extracts were done with an Agilent Technologies 7890A GC system equipped with a flame ionization detector (FID) set to 300 C and a VF-5ms column (CP8944; 30 m Â 0.25 mm, ID 0.25 mm). H 2 flow was set to 30 mL min À1 , Air Flow to 400 ml min À1 and N 2 flow to 30 mL min À1 . Injector temperature was set to 250 C and 1 mL of the sample was injected in splitless mode. Initial oven temperature was set to 110 C, held for 1 minute and increased afterwards to 200 C with a rate of 10 C/min followed by an increase to 300 C in steps of 25 C/min. The temperature of 300 C was held for 10 minutes.
For GC-MS measurements a Thermo Scientific Trace GC Ultra system with a ISQ mass spectrometer and a TG-SQC column (Thermo Scientific; 15 m Â 0,25 mm, ID 0,25 mm) was used. Injector temperature was set to 90 C and 1 mL of the sample was injected in splitless mode. Initial oven temperature was set to 60 C for 1 min, followed by an increase of the temperature to 200 C with a rate of 10 C/min. Afterwards the temperature was increased to 300 C with a rate of 25 C/min which was held for 10 minutes. Helium was used as a carrier gas with a flow of 0.7 ml min À1 .

1 H-NMR analysis
For NMR analysis, 20 mg of freeze-dried leaf material of the wild type and the nicotine-free plant or 10 mg of nicotine standard were mixed with 1 mL methanol-D4 and vortexed for one minute. After ultrasonication for 15 minutes the samples were centrifuged at 13,000 g for 5 minutes. Around 600 mL of the supernatant was filled into a 3 mm NMR-tube. 1 H-NMR measurements were done at 25 C and 600 MHz with the Bruker AV 600 Avance III HD (Cryoprobe) spectrometer. The data were analyzed using TopSpin 4.0.