The term new genomic techniques (NGTs), is an umbrella term to describe a variety of techniques that can alter the genetic material of an organism and that have emerged or have been developed since 2001, when the existing genetically modified organism (GMO) legislation was adopted. The analytical framework to detect GMOs in Europe is a established single harmonized procedure which is mandatory for authorization of GM food and feed, thus generating reliable, transparent and effective labelling scheme for GMO products. However, NGT products challenge the implementation and enforcement of the current regulatory system in the EU, relating in particular to the detection of NGT products that contain no foreign genetic material. Consequently, current detection methods will fail to meet minimum performance requirements. Although existing detection methods may be able to detect and quantify even small alterations in the genome, this does not necessarily confirm the distinction between products resulting from NGTs subject to the GMO legislation and other products. Therefore, this study provides a stepwise approach for the in silico prediction of PCR systems specificity by testing a bioinformatics pipeline for amplicon and primer set searches in current genomic databases. We have tested our methodology in two mutant genotypes produced by CRISPR/Cas9 in Arabidopsis thaliana. Overall, our results demonstrated that the single PCR system developed for the identification of a nucleotide insertion in the grf1-3 genotype has sufficient hits in the databases that would not allow discrimination of this mutanted event. Differently, the second mutated genotype grf8-61 which contains a -3bp deletion, found no matches in the sequence variant database. Our approach is the first step in decision making on PCR system and modification of them for empirical testings.

Profiling technologies, such as proteomics, allow the simultaneous measurement and comparison of thousands of plant components without prior knowledge of their identity. The combination of these non-targeted methods facilitates a more comprehensive approach than targeted methods and thus provides additional opportunities to identify genotypic changes resulting from genetic modification, including new allergens or toxins. The purpose of this study was to investigate unintended changes in GM Bt maize grown in South Africa. In the present study, we used bi-dimensional gel electrophoresis based on fluorescence staining, coupled with mass spectrometry in order to compare the proteome of the field-grown transgenic hybrid (MON810) and its near-isogenic counterpart. Proteomic data showed that energy metabolism and redox homeostasis were unequally modulated in GM Bt and non-GM maize variety samples. In addition, a potential allergenic protein – pathogenesis related protein -1 has been identified in our sample set. These finding highlight the suitability of unbiased profiling approaches to complement current GMO risk assessment practices worldwide.

CRISPR-Cas9 technology allows the modification of DNA sequences in vivo at the location of interest. Although CRISPR-Cas9 can produce genomic changes that do not require DNA vector carriers, the use of transgenesis for stable integration of DNA coding for gene-editing tools into plant genomes is still the most used approach and it can generate unintended transgenic integrations, while Cas9 prolonged expression can increase cleavage at off-target sites. In addition, the selection of genetically modified cells from millions of treated cells, especially plant cells, is still challenging. These downfalls can be avoided with the delivery of preassembled ribonucleoprotein complexes (RNPs) composed of purified recombinant enzyme Cas9 and in vitro- transcribed guide RNA (gRNA) molecules in a protoplast system. We therefore aimed to develop the first DNA-free protocol for gene-editing in maize and introduced RNPs into their protoplasts with PEG 4000. We performed effective transformation of maize protoplasts using different gRNAs sequences targeting the inositol phosphate kinase gene and applying two different exposure times to RNPs. Using low-cost Sanger sequencing protocol, we observed an efficiency rate of 0.85 up to 5.85%, which is equivalent to DNA-free protocols used in other plant species. A positive correlation was displayed between exposure time and mutation frequency. Mutation frequency was gRNA sequence- and exposure time-dependent. In summary, we demonstrated the suitability of RNP transfection as an effective screening platform for gene-editing in maize. This efficient and relatively easy assay method for selection of gRNA suitable for editing of gene of interest will be highly useful for genome editing in maize, since genome size and GC-content are large and high in maize genome, respectively. Nevertheless, the large amplitude of mutations at target site requires scrutiny when checking mutations at off-target sites and potential safety concerns.