Nicotine-free, nontransgenic tobacco (Nicotiana tabacum l.) edited by CRISPR-Cas9.

Julia Schachtsiek,Felix Stehle

doi:10.1111/pbi.13193

Abstract

Nicotine-free, nontransgenic tobacco (Nicotiana tabacum l.) edited by CRISPR-Cas9.

Highlights

We aimed for a simple CRISPR Cas9-based knockout strategy and searched for an identical target sequence present in all six published coding sequences (BBLa, BBLc, BBLd.2 originated from Nicotiana sylvestris; BBLb, BBLd.1, BBLe from Nicotiana tomentosiformis; Kajikawa et al, 2011, 2017) to enable the use of a single-guide RNA
For BBLa, the sequencing trace showed a double peak for thymine and guanine, which is consistent with the results from the cloning experiments, in which both base pair insertions were detected
Analysis of the GC measurements showed that the alkaloids anatabine, nornicotine and anabasine were present in wild-type extracts, but the latter two at the detection limit

Summary

Introduction

We aimed for a simple CRISPR Cas9-based knockout strategy and searched for an identical target sequence present in all six published coding sequences (BBLa, BBLc, BBLd.2 originated from Nicotiana sylvestris; BBLb, BBLd.1, BBLe from Nicotiana tomentosiformis; Kajikawa et al, 2011, 2017) to enable the use of a single-guide RNA. In order to identify nicotine-free tobacco plants carrying knockouts in all six BBL genes, progenies of plant T0 4 up to generation T3 were screened initially with regard to their nicotine content. The GC analysis of the nicotine content of the analysed T2 and T3 plants resulted in minimal peaks with retention times identical to the nicotine standard. Since the signal-to-noise ratio of the peaks was too low for an automated peak detection, a manual analysis of the peak area was performed for estimation of the residual nicotine content.

Results

Conclusion