The gene knock-out in N. benthamiana L. and P. somniferum L. by using RNA-guided CRISPR/Cas9 system

Genome modifications in plants have been doing for many centuries by mankind since from the times of ancient farmers to the scientists for now by applying selective breeding and GM techniques. Recently, a new synthetic non-GM technology has been developed for transcriptional-level manipulations. CRISPR system is an RNA-guided transient genome editing tool, that is generally composed of a target-specifically designed chimeric sgRNA and a Cas9 endonuclease, which forms DSBs in DNA and results a frame-shift mutation to knock-out any desired gene or genes in genome. CRISPR system employs genome repair mechanims of HR or NHEJ after introducing a cut into DNA and mediates genome editing by using organisms own repair mechanisms. Type II CRISPR/SpCas9 is a highly-specific and robust system which is a state of art among the other genome-editing technologies that is adapted to plant sicence. In this study, Opium poppy (Papaver somniferum L.), one of the most popular medicinal aromatic plants, and a well-known model organism Nicotina benthamiana L. genomes were investigated on the applicabilitiy of CRISPR/Cas9 system by using synthetic and viral based transcribed sgRNAs. sgRNAs targeting Pap4OMT2 and NbPDS genes were specifically designed and used to target those specific gene segments with SpCas9 endonuclease and formed InDels were demonstrated. Thus, the applicability of CRISPR/Cas9 system in non-model papaver species was shown for the first time in literature.