RESUMO
The term new genomic techniques (NGTs) is an umbrella term used 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 used to detect GMOs in Europe is an established single harmonized procedure that is mandatory for the authorization of GM food and feed, thus generating a reliable, transparent, and effective labeling scheme for GMO products. However, NGT products can 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, the current detection methods might fail to meet the 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. In addition, it also empirically tested the PCR system evaluated during the in silico analysis. Two mutant genotypes produced by CRISPR-Cas9 in Arabidopsis thaliana were used as a case study. Overall, our results demonstrate that the single PCR system developed for identifying a nucleotide insertion in the grf1-3 genotype has multiple matches in the databases, which do not enable the discrimination of this mutated event. Empirical assays further support this demonstration. In contrast, the second mutated genotype, grf8-61, which contains a -3 bp deletion, did not yield any matches in the sequence variant database. However, the primer sequences were not efficient during the empirical assay. Our approach represents a first step in decision making for analytical methods for NGT detection, identification, and quantification in light of the European labeling regulations.
RESUMO
CRISPR/Cas9-based ribonucleoprotein (RNP)-mediated system has the property of minimizing the effects related to the unwanted introduction of vector DNA and random integration of recombinant DNA. Here, we describe a platform based on the direct delivery of Cas9 RNPs to soybean protoplasts for genetic screens in knockout gene-edited soybean lines without the transfection of DNA vectors. The platform is based on the isolation of soybean protoplasts and delivery of Cas RNP complex. To empirically test our platform, we have chosen a model gene from the soybean genetic toolbox. We have used five different guide RNA (gRNA) sequences that targeted the constitutive pathogen response 5 (CPR5) gene associated with the growth of trichomes in soybean. In addition, efficient protoplast transformation, concentration, and ratio of Cas9 and gRNAs were optimized for soybean for the first time. Targeted mutagenesis insertion and deletion frequency and sequences were analyzed using both Sanger and targeted deep sequencing strategies. We were able to identify different mutation patterns within insertions and deletions (InDels) between + 5 nt and -30 bp and mutation frequency ranging from 4.2 to 18.1% in the GmCPR5 locus. Our results showed that DNA-free delivery of Cas9 complexes to protoplasts is a useful approach to perform early-stage genetic screens and anticipated analysis of Cas9 activity in soybeans.
