A Real-Time Quantitative PCR Method Specific for Detection and Quantification of the First Commercialized Genome-Edited Plant.

Pradheep Chhalliyil,Heini Ilves,Brian Johnston,Stephanie Howard,John Fagan,Sergei Kazakov

doi:10.3390/foods9091245

Pradheep Chhalliyil, Heini Ilves + Show 4 more

Open Access

https://doi.org/10.3390/foods9091245

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Abstract

Discussion regarding the regulatory status of genome-edited crops has focused on precision of editing and on doubts regarding the feasibility of analytical monitoring compliant with existing GMO regulations. Effective detection methods are important, both for regulatory enforcement and traceability in case of biosafety, environmental or socio-economic impacts. Here, we approach the analysis question for the first time in the laboratory and report the successful development of a quantitative PCR detection method for the first commercialized genome-edited crop, a canola with a single base pair edit conferring herbicide tolerance. The method is highly sensitive and specific (quantification limit, 0.05%), compatible with the standards of practice, equipment and expertise typical in GMO laboratories, and readily integrable into their analytical workflows, including use of the matrix approach. The method, validated by an independent laboratory, meets all legal requirements for GMO analytical methods in jurisdictions such as the EU, is consistent with ISO17025 accreditation standards and has been placed in the public domain. Having developed a qPCR method for the most challenging class of genome edits, single-nucleotide variants, this research suggests that qPCR-based method development may be applicable to virtually any genome-edited organism. This advance resolves doubts regarding the feasibility of extending the regulatory approach currently employed for recombinant DNA-based GMOs to genome-edited organisms.

Highlights

In recent years, biotechnologists have begun to employ genome editing methods such as ODM, CRISPR/Cas, TALEN and ZFN to modify the characteristics of organisms important in production of food and feed
Our work provides a definitive answer to this question, demonstrating that highly sensitive and specific PCR methods that meet GMO regulatory requirements can be developed to detect and quantify edited organisms even when the genome edit consists of only a single base pair alteration, and even in cases of multicopy gene targets
We have developed a sensitive, GMO regulation-compliant method for detecting the first genome-edited crop to be commercialized and suggest that it may represent a general approach for detecting genome-edited organisms

Summary

Introduction

Biotechnologists have begun to employ genome editing methods such as ODM (oligonucleotide-directed mutagenesis), CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR associated protein), TALEN (transcription activator-like effector nuclease) and ZFN (zinc finger nuclease) to modify the characteristics of organisms important in production of food and feed. The European Court of Justice has ruled [4] that crops modified by directed mutagenesis fall within the scope of Directive 2001/18/EC on the release of GMOs into the environment [5]. This means that genome-edited crops are regulated according to that Directive [5]. Preceding and following this decision, there has been sustained discussion with wide-ranging perspectives regarding the regulation of these methods among EU [6,7,8] and member state [9,10,11,12] government representatives, within the Convention on Biodiversity [13,14], and among representatives of the biotechnology industry [15,16] and the academic community [3,17,18,19,20,21,22,23]

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