Agronomy Publications Agronomy 2-2016 The Regulatory Status of Genome-edited Crops Jeffrey D. Wolt Iowa State University, [email protected] Kan Wang Iowa State University, [email protected] Bing Yang Iowa State University, [email protected] Follow this and additional works at: http://lib.dr.iastate.edu/agron_pubs Part of the Agricultural Science Commons, Agriculture Commons, Agronomy and Crop Sciences Commons, Genomics Commons, and the Plant Breeding and Genetics Commons The ompc lete bibliographic information for this item can be found at http://lib.dr.iastate.edu/ agron_pubs/95. For information on how to cite this item, please visit http://lib.dr.iastate.edu/ howtocite.html. This Article is brought to you for free and open access by the Agronomy at Iowa State University Digital Repository. It has been accepted for inclusion in Agronomy Publications by an authorized administrator of Iowa State University Digital Repository. For more information, please contact [email protected]. The Regulatory Status of Genome-edited Crops Abstract Genome editing with engineered nucleases (GEEN) represents a highly specific nda efficient tool for crop improvement with the potential to rapidly generate useful novel phenotypes/traits. Genome editing techniques initiate specifically targeted double strand breaks facilitating DNA-repair pathways that lead to base additions or deletions by non-homologous end joining as well as targeted gene replacements or transgene insertions involving homology-directed repair mechanisms. Many of these techniques and the ancillary processes they employ generate phenotypic variation that is indistinguishable from that obtained through natural means or conventional mutagenesis; and therefore, they do not readily fit current definitions of genetically engineered or genetically modified used within most regulatory regimes. Addressing ambiguities regarding the regulatory status of genome editing techniques is critical to their application for development of economically useful crop traits. Continued regulatory focus on the process used, rather than the nature of the novel phenotype developed, results in confusion on the part of regulators, product developers, and the public alike and creates uncertainty as of the use of genome engineering tools for crop improvement. Keywords engineered nucleases, site-specific utm agenesis, site-directed nucleases, homology-directed repair, CRISPR/ Cas9, TALEN Disciplines Agricultural Science | Agriculture | Agronomy and Crop Sciences | Genomics | Plant Breeding and Genetics Comments This article is from Plant Biotechnology Journal 14 (2015): 510–518, doi:10.1111/pbi.12444. Posted with permission. Rights This is an open access article under the terms of the Creative Commons Attribution-NonCommercial- NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made. This article is available at Iowa State University Digital Repository: http://lib.dr.iastate.edu/agron_pubs/95 Plant Biotechnology Journal (2016) 14, pp. 510–518 doi: 10.1111/pbi.12444 Review article The Regulatory Status of Genome-edited Crops Jeffrey D. Wolt1,2,3,*, Kan Wang1,3 and Bing Yang3,4 1Department of Agronomy, Iowa State University, Ames, IA, USA 2Biosafety Institute for Genetically Modified Agricultural Products, Iowa State University, Ames, IA, USA 3Crop Bioengineering Consortium, Iowa State University, Ames, IA, USA 4Department of Genetics, Development, and Cell Biology, Iowa State University, Ames, IA, USA Received 30 April 2015; Summary revised 24 June 2015; Genome editing with engineered nucleases (GEEN) represents a highly specific and efficient tool accepted 3 July 2015. for crop improvement with the potential to rapidly generate useful novel phenotypes/traits. *Correspondence (Tel +1 515 294 6899; Genome editing techniques initiate specifically targeted double strand breaks facilitating DNA- fax +1 515 294 2014; repair pathways that lead to base additions or deletions by non-homologous end joining as well email [email protected]) as targeted gene replacements or transgene insertions involving homology-directed repair mechanisms. Many of these techniques and the ancillary processes they employ generate phenotypic variation that is indistinguishable from that obtained through natural means or conventional mutagenesis; and therefore, they do not readily fit current definitions of genetically engineered or genetically modified used within most regulatory regimes. Addressing ambiguities regarding the regulatory status of genome editing techniques is critical to their application for Keywords: engineered nucleases, development of economically useful crop traits. Continued regulatory focus on the process used, site-specific mutagenesis, site-directed rather than the nature of the novel phenotype developed, results in confusion on the part of nucleases, homology-directed repair, regulators, product developers, and the public alike and creates uncertainty as of the use of CRISPR/Cas9, TALEN. genome engineering tools for crop improvement. Introduction differs from GEEN approaches in that OMM does not deliver a nuclease to the site of action. Optimized OMM trait development Genome editing with engineered nucleases (GEEN) has rapidly systems are resulting in the first genome-edited crops for emerged as a leading tool for investigating gene function and for commercial release (Pratt, 2012). creating genetic variation using site-directed genomic alterations. Recent discoveries and advances in genome editing use site- When applied to economically important plant species, GEEN directed nucleases (SDNs) where engineering of the nuclease additionally provides a highly specific and efficient means to allows for highly specific targeting to any given gene of interest. generate useful novel phenotypes. With the advent of genome The array of SDNs which have been used for genome editing in editing as a readily accessible technology to the research higher plants encompasses engineered meganucleases (EMNs community, the question arises as to how plants expressing also referred to as LAGLIDADG endonucleases or homing unique traits derived by genome editing will be received by the nucleases), zinc finger nucleases (ZFNs), transcriptional activator- public at large and treated within various regulatory domains. like effector nucleases (TALENs) and clustered regularly This question cannot be easily answered from a process interspaced short palindromic repeats in conjunction with the viewpoint, as site-directed genome editing may range from associated Cas9 endonuclease (CRISPR/Cas9) (Gaj et al., 2013; mutations involving a single base change to transgene insertions. Osakabe and Osakabe, 2014). These various GEEN methods Addressing ambiguities regarding the regulatory status of operate in a similar fashion to generate a double strand break genome-edited crops is critical to the application of genome (DSB) at a specific location in the genome and are comprised of editing for developing economically useful crop traits. In this engineered proteins consisting of a DNA binding domain to review, progress in GEEN and related techniques in higher plants confer site specificity and an endonuclease domain to cause the is considered with respect to the current regulatory status for DSB (Curtin et al., 2012). A variety of natural DNA repair genome-edited crops. mechanisms involving nonhomologous end joining (NHEJ) or homologous recombination (HR) can be exploited to allow for Genome editing techniques targeted genome modifications in vivo. When NHEJ rejoins DSBs to repair broken chromosomes, the result is often imprecise, The earliest example of genome editing (Table 1) in higher plants introducing mutations at the cut site that can alter gene function involved oligonucleotide-mediated mutagenesis (OMM) to cause and serve as a source of induced genetic variation. Alternatively, site-specific gene targeting using chemically synthesized oligonu- DSB-induced HR can be used for highly specific gene targeting cleotides with base replacement or addition caused by endoge- (homology-directed repair, HDR) involving either gene replace- nous DNA-repair enzymes (Beetham et al., 1999). The method ment or gene insertion enabling precise genome modification The copyright line for this article was changed on 8 October 2015 after original online publication. 510 ª 2015 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd. This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made. The Regulatory Status of Genome-edited Crops 511 Table 1 Genome editing acronyms, terms and definitions (Breyer et al., 2009; Kim and Kim, 2014; Osakabe and Osakabe, 2014; de Souza, 2012) CRSPR Clustered Regularly-Interspaced Short Programable nucleases comprised of bacterially derived endonuclease (Cas9) and a single-guide RNA (sgRNA) Paloindromic Repeats DSB Double Strand Break Cleavage in both strands of double-stranded DNA where the two strands have not separated EMN Engineered Mega Nuclease Microbially derived meganucleases that are modified, fused, or rationally designed to cause site-directed DSB. Also referred to as LAGLIDADG endonucleases or homing