Risk analysis for genome editing-derived food safety in China

Food Control 84 (2018) 128e137

Contents lists available at ScienceDirect

Food Control journal homepage: www.elsevier.com/locate/foodcont

Review

Risk analysis for genome editing-derived food safety in China Wei Gao a, Wen-Tao Xu a, b, Kun-Lun Huang a, b, Ming-zhang Guo a, Yun-Bo Luo a, b, * a

Beijing Advanced Innovation Center for Food Nutrition and Human Health, College of Food Science and Nutritional Engineering, China Agricultural University, Beijing, 100083, China b Key Laboratory of Safety Assessment of Genetically Modified Organism (Food Safety), Ministry of Agriculture, Beijing, 100083, China

a r t i c l e i n f o

a b s t r a c t

Article history: Received 4 May 2017 Received in revised form 23 July 2017 Accepted 24 July 2017 Available online 25 July 2017

Novel foods derived from genome-editing techniques call for an updated risk analysis of the use of plant produced by genome editing, especially in China. As a developing country with the largest population and limited arable land, China has invested intensively in genetically modified (GM) crops to improve agricultural productivity, which is aimed to secure food security and safety, the top priority for China. China has recently made great progress in genome-editing technology due to its powerful applications in the field of crop improvement such as rice and wheat. To ensure food safety and agricultural commodity trade, China has developed a regulatory system for the risk assessment and management of GM products, and the discussion on how to regulate products derived from genome-editing technology have also been initiated. A working group within National Biosafety Committee was established to provide technical assistance on risk assessment of new techniques including genome-editing. As GMO safety remains a public concern in China, genome-editing technology is in many ways even more precise and predictable than GM. From that perspective, genome-editing technology may be accepted by the public more easily with active communication with broad stakeholders, such as government, consumer, media, industry and others. This article reviews current developments in genome-editing technology and its applications in plant in China, regulatory status of genome editing-derived products around the world, and risk analysis framework for genome-editing derived food in China. © 2017 Elsevier Ltd. All rights reserved.

Keywords: Risk analysis Genome editing Food safety Regulatory framework

Contents 1. 2.

3.

4.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 129 Genome-editing technology development in China . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 129 2.1. Comparison of ZFN, TALEN, MegaN and CRISPR/Cas 9 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 129 2.2. Genome-editing technology development in plants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 130 2.3. Challenges for genome-editing technology in China . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 131 Regulatory status on genome-editing around the world and challenges for China . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 131 3.1. Situation in US, Canada and Argentina . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 131 3.1.1. Regulatory consideration for genome editing-derived products in the US . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 131 3.1.2. Regulatory framework for genome editing-derived products in Canada . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 132 3.1.3. Regulatory framework for genome editing-derived products in Argentina . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 133 3.2. Regulatory situation in EU . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 133 3.3. Challenges and the situation in China . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 133 Detection methods for genome editing-derived food and food labels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 133 4.1. Detection methods for GMOs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 134 4.2. Detection methods for genome-editing derived products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 134 4.3. Labeling system for genome editing-derived food in China . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 134

* Corresponding author. Beijing Advanced Innovation Center for Food Nutrition and Human Health, College of Food Science and Nutritional Engineering, China Agricultural University, Beijing, 100083, China. E-mail address: [email protected] (Y.-B. Luo). http://dx.doi.org/10.1016/j.foodcont.2017.07.032 0956-7135/© 2017 Elsevier Ltd. All rights reserved.

W. Gao et al. / Food Control 84 (2018) 128e137

5.

6.

129

A risk analysis framework in China for biotech-derived food that keeps pace with the times . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 134 5.1. Food safety is one of the Chinese government's top priorities . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 134 5.2. Risk management for GM food in China . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 135 5.2.1. Risk-management framework . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 135 5.2.2. Regulations for risk management . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 135 5.3. Risk assessment for GM food in China . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 135 5.4. Risk communication for GM food in China . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 135 5.5. Risk analysis for genome-editing derived products in China . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 136 Suggestions and perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 136 Conflict of interest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 136 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 136

1. Introduction

editing technology compared with GM.

Like the search/replace function in Microsoft Word, genomeediting technology makes it possible to accurately edit genomes. Genome-editing technology has been widely used in medicine, animals and agriculture. For example, it has been applied in medicine to cure genetic diseases such as sickle-cell anemia (Gaj, Gersbach, & Barbas, 2013), in animal husbandry for the cultivation of hornless dairy cows (Carlson, Lancto, Zang, Kim, Walton, & Oldeschulte, 2016) and more widely in agriculture to produce disease-resistant rice (Li, Liu, Spalding, Weeks, & Yang, 2012), higholeic soybeans (Haun, Coffman, Clasen, Demorest, Lowy, & Ray, 2014) and anti-browning mushrooms (Waltz, 2016a, 2016b), which could reduce food waste. As a developing country with the largest population, China is making efforts to employ genetic-engineering technology to boost agricultural productivity even there are some challenges for adopting GM crops in developing world (Aghaee, Olkowski, Shelomi, Klittich, Kwok, & Maxwell, 2015). Chinese government has steadily increased the investment in agricultural since hightech 863 program in 1986. In 2008, China launched the National Genetically Modified Organism (GMO) New Variety Breeding Program, one of the 16 National Science and Technology Major Projects from 2006 to 2020 (Li, Peng, Hallerman, & Wu, 2014). Since 2007, the No.1 Central Document issued by Central Committee of the Communist Party of China and the State Council has mentioned biotechnology 8 times, which requests to increase public awareness of and promote biotechnology. With more than 20 years of promotion of agricultural biotechnology, China has also developed a regulatory system for risk assessment and management of GM products to ensure food safety. Based on those intensive investments, many new genetically modified (GM) crop traits and varieties have been developed in China with characteristics such as insect resistance, herbicide tolerance, and stress tolerance. However, only insect-resistant Bt cotton and disease-resistant papaya have been commercialized on a large scale (Li, Peng, Hallerman, & Wu, 2014). Biosafety certificates for commercial planting for two GM Bt rice lines and one GM phytase corn were issued in 2009 and renewed in 2015, but cultivation has not yet occurred. In contrast to conventional genetic modification resulting from the insertion of large pieces of exogenous DNA, genome-editing techniques generate phenotypic variation that is indistinguishable from that generated by natural means or induced mutations. Given the debate and concern on GM food safety, it is important to get clarity on the regulatory status of genome-editing, which is the key to genome-editing techniques’ application (Jones, 2015). With clear and science based regulatory path, it is possible for China to adopt the genome editing derived products easier because of less concerns and more advanced research and development in genome

2. Genome-editing technology development in China Genome-editing techniques with sequence-specific nucleases (SSNs) creates DNA double-strand-breaks (DSBs) in the genomic target sites that lead to gene mutations, insertions, replacements or chromosome rearrangements by non-homologous end joining (NHEJ) or homology-directed repair (HR) mechanisms. NHEJ produces small insertions or deletions (indels) and is useful for disrupting gene function. HR can induce precise gene repair of one of thousands of base pairs in the presence of a homologous donor molecule to correct point mutations and introduce exogenous sequences (Fig. 1). There are four major SSNs: meganucleases, or homing endonucleases (HEs), zinc finger nuclease (ZFN), transcription activator-like effector nuclease (TALEN) and the clustered regularly interspaced short palindromic repeats/CRISPR-associated
noret, Fontanie re, 9 (CRISPR/Cas 9) system (Gaj et al., 2013; Me
my, 2013; Smith, Grizot, Arnould, Jantz, Tesson, Thinard, & Re Duclert, Epinat, & Chames, 2006). As Chinese scientists adopted genome editing as a powerful tool for crop improvement, functional genomics and drug discovery very quick, Chinese publications and patents in genome editing technology have been ranked 2nd in the world (Zhou, 2016). 2.1. Comparison of ZFN, TALEN, MegaN and CRISPR/Cas 9 ZFN, the first genomic-editing strategy, uses custom DNA endonucleases that are mostly based on the Fokl restriction enzyme fused to a zinc finger DNA-binding domain engineered to target a specific DNA sequence. But researchers must order reagents from Sigma Aldrich as ZFN technology is licensed to Sigma Aldrich for research applications by Sangamo Biosciences, the owner of this technology (Perkel, 2013), which makes zinc fingers relatively expensive. In addition, off-target effects created by site-specific nucleases are high and can be toxic to cells. Similar to ZFNs, TALENs induce targeted DSBs that activate DNA damage response pathways and enable custom alterations. (Gaj et al., 2013). TALENs can perform most of the functions of ZFNs but provide high specificity and a low chance of off-target effects. However, the large size of TALENs may limit their delivery by sizerestricted vectors, and construction is more complicated (Shan & Gao, 2015). Mega Nucleases (MegaN) recognise DNA sequences of 12e40 bases by DNA-protein interaction, and contain nuclease activities. The recognition sites of natural MegaN are limited, while engineered MegaN could be constructed by introducing amino acids
noret et al., 2013; into recognition domain to create new MegaN (Me Smith et al., 2006). MegaN could be used in genome editing with high specificity and less toxicity to the cell. However, similar to

130

W. Gao et al. / Food Control 84 (2018) 128e137

Fig. 1. Overview of DNA break repair mechanisms for genome editing.

TALENs, the construction of engineered MegaN is complicated which limits its application. In the CRISPR/Cas 9 system, RNA-guided DNA endonucleases cleave a target sequence. The Cas9 protein is directed toward the target site using a single guide RNA (sgRNA) consisting of a 20-base pair protospacer that attaches to the complementary strand of the target sequence and a constant region interacting with the Cas 9 protein. Comparing with the traditional ZFN, TALEN, and MegaN approaches, CRISPR/Cas technology is more straightforward and affordable for genome-editing because specificity is dictated by DNA complementarity without the need for multistep protein engineering (Shan & Gao, 2015). The mechanism of these four kinds of genome-editing nucleases is as below (Fig. 2) A, Mega Nuclease (MegaN) recognizes DNA sequences of 12e40 bases by DNA-protein interaction, and contains nuclease activities. B, Zinc Finger Nuclease (ZFN) contains several ZFN domains, with each ZFN domain recognizes 3 bases by DNA-protein interaction. FokI heterodimers are formed with nuclease activities when the recognition sites two ZFN are at a distance of 6e8 bp in different strands. C, Transcription Activator-like Effector Nuclease (TALEN) contains several TALE domains with each TALE domain recognizes 1 base by DNA-protein interaction. The formation of nuclease activities by FokI heterodimers is the same with that in ZFN. D, CRISPR-Cas9 System recognizes DNA sequences by combination of sgRNA, which make it much easier to construct than DNA-protein interaction based genome-editing nucleases. 2.2. Genome-editing technology development in plants In the past few years, the development of genome-editing technology in China has progressed rapidly, with powerful applications in plants. CRISPR/Cas system is much easier to implement comparing with ZFNs and TALENs and MegaN, as only sg-RNA must be customized to target the genes of interest. (Shan, Wang, Li, & Gao, 2014). Three leading Chinese scientists published their studies demonstrating

CRISPR/Cas genome-editing in crops in 2013, representing the first reports in the world: Gao Caixia and her team from the Institute of Genetics and Developmental Biology at Chinese Academy of Sciences, Zhu Jian-Kang and his team from the Shanghai Center for Plant Stress Biology at Chinese Academy of Sciences, and Qu Li-Jia and his team from Peking University. In 2013, Gao Caixia and her team's study showed that a customizable sgRNAcan direct Cas 9 to induce sequence-specific genome modifications in the two most widely cultivated food crops, rice and common wheat, using CRISPR/Cas for targeted mutagenesis of the OsPDS gene in rice and TaMLO gene in wheat(Shan, Wang, Li, Zhang, Chen, & Liang, 2013). Subsequently, in 2014, Gao Caixia & Jin-Long Qiu reported that TALEN-induced mutation of all three TaMLO homeologs in wheat confers heritable broad-spectrum resistance to powdery mildew, and they used the CRISPR-Cas 9 tool to generate transgenic wheat plants carrying mutations in the TaMLO-A1 allele (Wang, Cheng, Shan, Zhang, Liu, & Gao, 2014). In the same year, Gao Caixia and her team's study also showed that both TALENs and the CRISPR/Cas system can be used for genome modification in maize. They reported targeted mutagenesis in Zea mays using TALENs and the CRISPR/Cas system. Five TALENs targeting 4 genes were designed, and targeting efficiencies of up to 23.1% in protoplasts were obtained. Approximately 13.3%e39.1% of the transgenic plants had somatic mutations. They also found the CRISPR/Cas system induced targeted mutations in Z. mays protoplasts with efficiencies (13.1%) similar to those obtained with TALENs (9.1%) (Liang, Zhang, Chen, & Gao, 2014). In studies in 2015 of a geminivirus, beet severe curly top virus (BSCTV), Gao Caixia's team reported that transient assays performed in Nicotiana benthamiana demonstrated that sgRNACas 9 constructs inhibited virus accumulation and introduced mutations at the target sequences (Ji, Zhang, Zhang, Wang, & Gao, 2015). In 2016, this team reported the use of the two genomeediting methods to edit genes in hexaploid bread wheat and tetraploid durum wheat and showed that the mutants were generated with no detectable transgenes(Zhang, Liang, Zong,

W. Gao et al. / Food Control 84 (2018) 128e137

131

editing in plants in China (Fig. 3). 2.3. Challenges for genome-editing technology in China

Fig. 2. Mechanism of four kinds of genome-editing nucleases.

Wang, Liu & Chen, 2016). In 2017, Gao Caixia's team used CRISPR/ Cas 9 nickase-cytidine deaminase fusion to achieve precise base editing in rice, wheat and maize without the need for a foreign DNA donor or double-stranded DNA (Zong, Wang, Li, Zhang, Chen, & Ran, 2017). Zhu Jian-Kang and his team demonstrated highly efficient targeted mutagenesis in multiple genes in Arabidopsis and rice using engineered CRISPR/Cas. The Cas 9 gene was driven by the CaMV35S promoter and a sgRNA was driven by the AtU6-26 promoter in Arabidopsis or the OsU6-2 promoter in rice. The results of the study demonstrated that the engineered CRISPR/Cas was active in creating DSBs at target sites of the plant genome, which can be employed to achieve targeted genome modifications in both dicot and monocot plants. It also means genome editing technology will make targeted gene editing a routine practice in both model plants and crops (Feng, Zhang, Ding, Liu, Yang, & Wei, 2013; Mao, Zhang, Xu, Zhang, Gou, & Zhu, 2013). Qu Li-Jia and his team also showed that transgenic rice with mutations in specific genes could be generated using CRISPR/Cas technology in a straightforward manner (Miao, Guo, Zhang, Huang, Qin, & Zhang, 2013). In addition to the progress made in rice, wheat and maize, some studies using CRISPR/Cas system in Populus (Fan, Liu, Li, Jiao, Li, & Hou, 2015) and Tobacco (Gao, Wang, Ma, Xie, Wu, & Zhang, 2015) in China were reported in 2015. Below please find timeline figure covering chronological aspects of CRISPR/Cas mediated genome-

As transformative tools, ZFNs, TALENs, MegaN and Cas 9 nucleases could potentially revolutionize biological and medicine research. However, the concept of editing human DNA has often been controversial, and there was debate when the Chinese scientist Lu You, an oncologist at Sichuan University's West China hospital in Chengdu, planned to start testing cells modified with CRISPR on patients with lung cancer in August 2016 (Cyranosk, 2016). In contrast to medical and clinical research, genome editing in plants does not involve ethical issues and thus is more suitable for applied research. As genomes offer a lot of possible biding sites for a nuclease with an intrinsic potential to bind DNA, risk of off-target always remains. It is another challenge for the application of genome-editing technology as it has the potential to create cells with functional impairment, and how to evaluate the specificity of ZFN, TALEN, and Cas9 nucleases remains a concern (Cox, Platt, & Zhang, 2015). It is one of major concerns for applications of genome-editing techniques, especially for therapeutic and clinical application. To minimize off-target events, several measures should be taken into consideration such as choice of nuclease, target site choice and nuclease delivery method. ZNFs and TALENs are more specific but relatively more complex to product compared with CRISPR/Cas system. Recent studies have demonstrated minor modifications can increase the specificity and efficiency of CRISPR/Cas9 system, such as truncated guide RNAs (less than 20bp) (Fu, Sander, Reyon, Cascio, & Joung, 2014; Paul and Qi, 2016). In addition, it has been reported that few off-target sites in plant revealed by whole genome sequencing analysis, and these can be eliminated through backcrossing (Feng, Mao, Xu, Zhang, Wei.,Yang, 2014; Samanta, Dey, & Gayen, 2016; Zhang, Zhang, Wei, Zhang.,Guo, & Feng, 2014). Lastly, although China is the second-ranked country for publications on genome editing worldwide, original technology and patents in China are far fewer than those in EU and United States. In addition, there is a lack of overall strategic planning for genomeediting R&D, and thus core innovation is also far behind that in developed countries (Zhou, 2016). 3. Regulatory status on genome-editing around the world and challenges for China Genome-editing techniques especially CRISPR/Cas are emerging and regulatory environment for certain application of these techniques is evolving, and is the subject of a great deal of discussion globally. Like GM food, which mainly referring to the food derived from GM plants, genome-editing derived food is mainly referring to the food derived from plants produced using genome editing. From that point of view, it's important to have a quick overview on current global regulatory status on genome editing plant, which will help to understand where genome-editing derived foods are, and whether it will be differentiated from GM food. 3.1. Situation in US, Canada and Argentina 3.1.1. Regulatory consideration for genome editing-derived products in the US The USDA is the leading US regulator under the coordinated framework and provides guidance on how to regulate modern biotechnology products to technology developers. Based on the most update guidance, site-directed approaches that result in targeted deletions of endogenous nucleotides (SDN1) and approaches initiated with transgenesis that select for the absence of the

132

W. Gao et al. / Food Control 84 (2018) 128e137

Fig. 3. List of CRISPR/Cas mediated genome editing in plants in China.

transgenic elements in intermediate steps would not be regulated. However, site-directed methods involving targeted oligonucleotide insertions or substitutions will be evaluated on a case-by-case basis (Camacho, Deynze, Chi-Ham, & Bennett, 2014; Wolt, Wang, & Yang, 2016). The Animal and Plant Health Inspection Service (APHIS) at USDA informed Dow AgroSciences of Indianapolis that it would not regulate an herbicide-tolerant corn generated using zinc-finger nucleases in 2010 (Ledford, 2013). In April 2016, the USDA decided that a mushroom genetically modified with the geneediting tool CRISPR-Cas9 will not be regulated. By knocking out one of six genes for polyphenol oxidase (PPO), an enzyme that causes mushroom browning, a non-browning mushroom was obtained. In addition, a high amylopectin waxy corn developed by DuPont using CRISRP/Cas9 to knockout the endogenous waxy gene Wx1, which encodes the endosperm's granule-bound starch synthase responsible for making amylose, can be cultivated without oversight by the USDA (Waltz, 2016a, 2016b). FDA's consideration is focused on product-base, and holds the position of considering foods and feeds derived from genetic engineering technology as safe as their conventional counterparts following voluntary consultation process. Regulation related to genetic engineering technology within EPA is mainly focused to insect resistant traits. FDA announced a request for comments on January 18, 2017 to help inform its regulatory approach to foods derived from plants using genome editing, and it is extending the comment period to continue seeking scientific evidence and other input, which will be close on June 19, 2017 (Federal Register, 2017). APHIS at USDA is also proposing to revise the biotechnology

regulation at 7 Code of Federal Regulations (CFR) part 340 and open for public comments from January 19, 2017 until June 19, 2017, which is the first comprehensive revision of the regulation since they were established in 1987. Three categories of genetically engineered organisms would be excluded from the definition of genetic engineering including solely deletion or single base pair mutation, and insertion of donor nucleic acid which is not passed to recipient's genome and has not altered the DNA sequence of the progeny (USDA, 2017a,b).

3.1.2. Regulatory framework for genome editing-derived products in Canada The Canadian Regulatory Framework for Biotechnology, the basis of Canada's regulation of biotechnology, is triggered primarily by the “product” and its novel trait, not the process by which it was obtained (Schuttelaar and Partners, 2015). The definition of a novel trait is given by the Canadian Food Inspection Agency (CFIA) (Schuttelaar and Partners, 2015), where plant with a novel trait (PNT) developed from conventional breeding, mutagenesis, transgenesis or genome editing will all be subject to a similar regulatory approval process and are regulated by the Canadian Food Inspection Agency in cooperation with Health Canada. The sale of foods derived from these PNTs is controlled by Health Canada via the mandatory pre-market notification requirement (Wolt, Wang, & Yang, 2016). An herbicide-tolerant variety of canola developed by Cibus Global was approved for use in Canada by the Canadian Food Inspection Agency and Health Canada in March 2014. It is the first commercial crop generated using genome editing (Jones, 2015).

W. Gao et al. / Food Control 84 (2018) 128e137

3.1.3. Regulatory framework for genome editing-derived products in Argentina Thus far, Argentina is the only country in the world, which has issued specific regulation for the products derived from gene editing and other new breeding techniques (NBTs) (Whelan & Lema, 2015). Resolution no. 763/11 is the overall regulatory framework for GMOs; Resolution no. 701/11 details specific procedures for plant GMOs, including field trials and commercial release; and Resolution no. 173/15 of the Secretariat of Agriculture, Livestock and Fisheries describes the procedures for establishing if a crop obtained with the aid of new plant-breeding techniques is a GMO. The definition of GMO for Resolution no. 173/15 is same as in the Cartagena Protocol for LMOs. National Commission Advisor in Agricultural Biotechnology (CONABIA) shall perform the assessment for each NBT-derived crop submitted by applicants to see if the result of the breeding process is a new combination of genetic material. Based on the assessment roadmap in Argentina (Whelan & Lema, 2015), if genetic modification of the plant genome does not have sufficient entity to be considered a novel combination of genetic material, and if the information confirms that the temporary transgene was removed from the crop to be commercialized, CONABIA will inform the applicant that the crop is not covered by the GMO Resolution. But if the temporary transgene is not removed from final product, it will fall under regulatory framework for GMO. And if there is a new combination of genetic material, then it will also be regulated by GMO Resolution. If a hypothetical expected product derived from new plant breeding techniques still in the design stage, the applicants can submit a preliminary inquiry for whether this would be considered GMO. And the preliminary assessment and indicative answer shall be conducted and provided by CONABIA after receiving the inquiry. A few applications of the Early Consultation Procedure have been presented to consider whether a product obtained by NBT would be considered GMO or not in Argentina (especially SDN1 and SDN2). All were considered not to be GMO. 3.2. Regulatory situation in EU The European Academies Science Advisory Council (EASAC) concluded in 2013 that “the trait and product, not the technology, in agriculture should be regulated, and the regulatory framework should be evidence-based”, which was also endorsed by the academies. EASAC also asked EU regulators to confirm that the products of new breeding techniques do not fall in the scope of GMO legislation if they do not contain foreign DNA (Sprink, Eriksson, Schiemann, & Hartung, 2016). European Food Safety Authority (EFSA) established the new techniques working group in late 2007 and analyzed 8 new techniques including oigonucleotide directed mutagenesis (ODM), ZFN, cisgenesis, grafting, agro-infiltration, RNA-dependent DNA methylation (RdDM), reverse breeding and synthetic genomics in the context of GMO regulation. Since then, EFSA has issued scientific opinion addressing the safety assessment of plants developed through cisgenesis and intragenesis, Zinc Finger Nuclease 3 and other Site-Directed Nucleases in January and October 2012 respectively (EFSA, 2012a; EFSA, 2012b). From the scientific opinion, GMO regulation is applicable for plants developed through cisgenesis and intragenesis, and ZFN-3 technique. In 2015, per the request from the European Commission, the European Food Safety Authority (EFSA) stated in a letter that currently available ODM, ZFN-1 and -2 and similar SDN techniques create point mutations similar to those introduced through natural or induced mutagenesis and can thus be considered a form of mutagenesis (Sprink et al.,

133

2016). However, several NGOs released a legal analysis of Directive 2001/18/EC as strictly process-based. They strongly advised organisms produced by all new techniques (including ODM) should fall into the scope of GMO regulation based on that analysis. As of November 2016, the legal interpretation of genomeediting techniques and resulting crops in EU by the European Commission was still pending. 3.3. Challenges and the situation in China China has developed a regulatory system for risk management and assessment of biotech-derived products to ensure GM food safety since 1990s, which is conducted on a case-by-case basis. According to the “Regulation on Administration of Agricultural Genetically Modified Organisms Safety” (Regulation)issued by the State Council in 2001, Agricultural GMOs refer to animals, plants, microorganisms and their products whose genetic structures have been modified by genetic engineering technology for use in agricultural production or processing. From this point of view, genome editing derived products will fall in the scope of this Regulation. However, GMOs remain heavily regulated in many countries, including China, and the preparation of such regulatory packages is very expensive (approximately US $ 35 million per event) and timeconsuming (approximately 5.5 years on average for approval), which has significantly affected innovation and commercialization (Voytas and Gao, 2014), which China cannot afford as a developing country. Even in EU, many public researchers are seriously concerned about the situation in the EU regarding current legislation for GMO. In contrast to GMOs, the regulation for genome-edited products could be simplified, such as the USDA decision in 2016 not to regulate an anti-browning mushroom and a waxy corn genetically modified with the gene editing tool CRISPR/Cas 9, which would reduce by millions the cost of crop development (Waltz, 2016a). China is increasing research efforts in genome-editing techniques as an opportunity to accelerate speed-to-market with new traits, and Chinese scientists such as Gao Caixia and her team have made good progress on R&D and applications for genome-editing techniques. So it's important to have the clarity on regulatory because uncertainties may have negative impacts on research and innovation. China has initiated discussions on how to regulate products derived from genome-editing technology since 2015. A working group within the National Biosafety Committee (NBC) was established in September 2016 to provide technical assistance on how to regulate new techniques including genome editing in China, but no formal regulations have been issued yet. In addition, genome-editing technology is in many ways even more precise and predictable than GM, especially CRISPR/Cas 9, in which targeted single-nucleotide substitutions can be made without either double-strand breaks or a foreign DNA donor (Zong et al., 2017). From that perspective, genome-editing technology may be accepted by the public more easily than GMO, which remains a public concern in China. The comparative table for the regulatory status about genomeediting derived plants among the different countries is as below (Table 1). 4. Detection methods for genome editing-derived food and food labels As required in regulatory framework for risk assessment of GMOs in China, availability of detection method need to be considered for risk analysis of biotech derived food safety.

134

W. Gao et al. / Food Control 84 (2018) 128e137

Table 1 Regulatory status on genome-editing around the world. Country Argentina

Canada

China

EU

United States

Regulatory approach Regulation

Case-by-case exemption Resolution no. 173/15

Product-based: case-by-case, irrespective of process Current regulatory framework for biotechnology

Case-bycase Under review

Process-based

USDA: case-by-case FDA: voluntary process Currently under review

Examples

Not regulated as GMO

ODM canola was regulated as novel trait

No example

4.1. Detection methods for GMOs GMOs can be detected by using chemical analysis to detect the product of the enzymatic reaction, the new transgenic DNA that has been inserted or the new protein that has been expressed. In general, there are two specific approaches for detecting GMOs. One, polymerase chain reaction (PCR), is a DNA-based approach based on the detection of novel DNA sequences inserted into the genome. The other, enzyme-linked immunosorbent assay (ELISA), is a protein-based approach. During detection, sample preparation and Certified Reference Materials (CRMs) are critical and fundamental for both DNA-based and protein-based methods (Querci, Jermini, & Eede, 2006; Host-Jensen, Bertheau, Loose, Grohmann, Hamels, & Hougs, 2012). Numerous PCR-based methods have been developed that can detect and quantify GMOs in agricultural food and feed crops, including screening PCR, gene-specific PCR, construct-specific PCR, and event-specific PCR, all of which require small complementary DNA pieces as primers designed to hybridize to complementary sequence recognition sites on opposite strands of the gene of interest (Luo, 2016). It's concluded that DNA is the best target molecule for unambiguously detecting a change in the genetic material of plants, and PCR-based analytical method is the most commonly used technique for GMO detection, which provides higher sensitivity and specificity than protein-based approach. Protein-based detection methods will only be useful where a novel or changed protein is created by the genetic modification or protein product is removed (Lusser, Parisi, Plan, & Rodriguez-Cerezo, 2011). 4.2. Detection methods for genome-editing derived products As genome-editing has become a widely used tool, developing detection methods for genome editing-derived products and evaluating genome-editing tools are critical. However, genome editing produces small insertions or deletions, which complicates the detection of genome-editing events, especially single-nucleotide substitutions. Even with some prior information on the introduced modification is available, DNA-based methods would be possible for detection, but not possible to identify the genetic modification for ZFN-1 and ZFN-2 products because it cannot be distinguished at molecular level from products developed through mutagenesis (Lusser, Parisi, Plan, & Rodriguez-Cerezo, 2011). Sanger sequencing and gel-based methods lack the sensitivity required to detect such events in non-clonal cell populations. Nextgeneration sequencing (NGS) is expensive and time consuming, and the sample preparation can introduce bias. Berman et al. (Berman, Cooper, Zhang, Karlin-Neumann, Litterst, & Jouvenot, 2015) developed a method to detect CRISPR- and TALEN-mediated edits present at frequencies of less than 0.5%. Miaoka et al. established a method using droplet digital polymerase chain reaction (ddPCR) to detect

EU Court of Justice's decision on regulatory status expected in early 2018 ODM canola was not regulated as GMO non-browning mushroom and a high amylopectin waxy corn were not in Sweden, Finland, UK, Spain, Germany, Belgium, regulated as GM Netherlands, but regulated as GMO in Czech Republic

single-base mutations produced by genome editing efficiently and quantitatively (Miyaoka, Chan, & Conklin, 2016). 4.3. Labeling system for genome editing-derived food in China GM food labeling system is being debated and remains controversial in the world. There are more than 60 countries/regions have established regulations for labeling of GM food (Zhuo, 2014), in some of these the GM labeling is mandatory (Vilijoen & Marx, 2013), while some countries/regions are on voluntary basis. And some studies have been conducted to explore the impact of different GM food labeling system on consumer welfare and con
n ~ ez, & Alonso, sumers’ willingness to by GM food (Entrena, Ordo 2016; Zhao, Gu, Yue, & Ahlstrom, 2013). GM food labeling has also become issue in United States as well even it is the largest country adopted GM crop (Marchant & Cardineau, 2013). In July, 2016, President Barack Obama signed a bill requesting the United States Department of Agriculture (USDA) to establish a national disclosure standard for bioengineered foods within next two years. The National Bioengineered Food Disclosure Standard requires a national mandatory system for disclosing the presence of GMO ingredients through text labels, symbols, or digital links, such as QR codes (USDA, 2017a,b). In China, “Measures on Labeling Administration of Ag GMOs” released by the Ministry of Agriculture (MOA) in 2002 is a principle and catalogue for mandatory labeling. According to this regulation, 17 products derived from 5 biotech crops need to be labeled as zero tolerance, which is impracticable. The 5 crops are soybean, corn, rape, cotton and tomato (MOA, 2002). These guidelines have not been revised yet since 2002 even though there is no longer a biotech tomato on the market. Research on genome-editing technology is very popular in China, and the application of genome-editing technology in rice (Ji, Zhang, Zhang, Wang, & Gao, 2015), wheat (Zhang, Liang, Zong, Wang, Liu, & Chen, 2016), and sweet orange (Jia and Wang, 2014) has been reported, thus moving beyond the catalogue. With the rapid adoption of technology, the introduction of genome-edited crops and products on the market will increase with technology development. In addition, products derived from genome editing cannot be distinguished from those generated by natural or induced mutation. It is time to reconsider these regulations to implement a scientific and reasonable labeling system that is suitable for China. 5. A risk analysis framework in China for biotech-derived food that keeps pace with the times 5.1. Food safety is one of the Chinese government's top priorities As food safety is closely related to human life and health,

W. Gao et al. / Food Control 84 (2018) 128e137

economic development and social harmony, the Chinese government emphasizes food safety as a top priority. In the early 21st century, a food safety issue involving melamine-contaminated powdered milk occurred in 2008, and some food safety issues even emerged as a trade concern (Cicia, Caracciolo, Cembalo, Teresa, Grunert, & Krystallis, 2016; Luo, 2016), which not only damaged public health but also caused serious international concerns. The Chinese government has since placed great emphasis on improving the food safety regulatory system. The Food Safety Law was released in 2009 and revised in 2015 to ensure food safety and guarantee the health and safety of the public (NPC, 2015). In 2011, the China National Center for Food Safety Risk Assessment was established to provide technical support for the risk assessment of food safety. The China Food and Drug Administration (CFDA), which is directly under the State Council, were formed in 2013 to provide overall supervision of food safety. In 2016, CFDA issued 12 regulations and nearly 20 important rules to implement the Food Safety Law. Since 2013, the National Health and Family Planning Commission (NHFPC) has revisited and integrated food standards, and 926 food standards have been jointly released so far. As mentioned previously, genome-editing is falling in the current scope of regulation for GMOs in China because agricultural GMOs refer to animals, plants, microorganisms and their products whose genetic structures have been modified by genetic engineering technology for use in agricultural production or processing based on the Regulation issued by the State Council in 2001. Therefore, the general principle of risk analysis for GM food including risk assessment, management and communication is applicable for genome-editing derived food as well in China, but whether the similar risk assessment approach is necessary for genome-editing derived food needs further discussion on case-bycase basis. 5.2. Risk management for GM food in China China has the largest population in the world and is ranked eighth in terms of the global area of biotech crops, with 2.8 million hectares per the ISAAA annual report (ISAAA, 2016). To ensure the safe use of agricultural biotech products, biosafety management is required as an important part of the regulatory oversight of such products. With more than 20 years of nationwide promotion of agricultural biotechnology, a relatively welldeveloped risk-management system has been established. 5.2.1. Risk-management framework An inter-ministerial conference consisting of individuals from the departments of agriculture, science and technology, environmental protection, public health, foreign trade and economic cooperation, inspection and quarantine, and other relevant departments and led by the MOA is responsible for the overall strategy of agricultural biotechnology safety (Luo, 2016). As the leading agency, the MOA is responsible for safety assessment, supervision and management, drafting regulations and technical standards, import approval and labeling of agricultural biotechnology products. The National Biosafety Committee (NBC), which comprises 75 experts covering areas of agriculture, medicine, food, environment and detection, is responsible for technical review of safety assessment and consulting on agricultural biotechnology products (MOA, 2016). In total, 42 detection centers, including food safety detection, environmental safety detection, and characterization, have also been established to ensure the biosafety of such products (Luo, 2016).

135

5.2.2. Regulations for risk management The State Council issued the Regulation on Administration of Agricultural Genetically Modified Organisms Safety in 2001 as the general principle for risk assessment and management of agricultural GMO (State Council, 2001). Under this document, the MOA issued three lower-level regulations in 2002, including Measures on the Administration of Ag GMOs Import, Measures on the Safety Evaluation Administration of Ag GMO (revised in 2016), and Measures on Labeling Administration of Ag GMO. In addition, MOA released Measures on Examination and Approval for Processing GMOs in 2006. Measures on Quarantine Administration of GMO Product Import/Export was issued by the General Administration of Quality Supervision, Inspection and Quarantine (AQSIQ) in 2004. Thus far, 153 technical standards concerning GMO safety management has been established (Luo, 2016). 5.3. Risk assessment for GM food in China The objective of risk assessment is to identify the potential for GM food to have adverse effects on human health. Under the concepts and principles developed by OECD, FAO/WHO and Codex (FAO, 2008), China has developed its own biotech-derived food safety risk-assessment approach that is based on substantially equivalent, risk-based methods and conducted on a case-by-case basis. Specifically, biotech-derived food safety risk assessment in China mainly focuses on the following aspects (MOA, 2017). i) Description of Ag GMO and products including safety of donor and recipient organism, genetic modification, transformation process, construction and safety of the target gene and vector, inserted or deleted sequence, and expression data for the insert sequence. ii) Toxicological assessment of the protein expressed in transgenic plants, including information on the newly expressed protein, toxicological tests of the newly expressed protein, assessment of newly expressed non-protein substances and estimation of intake amount. iii) Analysis of key components including nutrients, natural toxins and harmful substances, anti-nutrients, other components and unexpected components. iv) Nutritional assessment including intake and utilization efficiency of key nutrients in animals, information on the levels of human nutrient intake, and assessments of the effects of the maximal possible intake level on the human diet. In addition, allergenicity assessment, whole food safety assessment, impact assessment of processing activities on safety and other safety assessments on a case-by-case basis are also needed. 5.4. Risk communication for GM food in China The risk analysis framework consists of three interconnected components: risk assessment, risk management and risk communication (Fig. 4) (WHO/FAO, 2016). As an essential part of the risk analysis, risk communication includes risk management decision-making processes, responsive consultation process and transparent safety assessment, which is extremely important to support information exchange among stakeholders. Stakeholders refer to government officials, scientific communities, general consumers, industry, media reporters and others. Effective risk communication will enable people to make decisions and facilitate mutual understanding among stakeholders, so that risk assessment and management can be enhanced

136

W. Gao et al. / Food Control 84 (2018) 128e137

Risk Management

Risk Assessment Process used to characterize risk,

ensure an appropriate

mutagenesis, should not follow the same safety assessments process for GMOs. Excessive safety assessments of new techniques, that neither improves the safety nor quality of crop products, will discourage investment and limit use of innovative techniques in the development of new varieties by the developers. (ILSI, 2017) From risk communication perspective, genome-editing technology looks different from conventional transgenic technology, which may be accepted by the public more easily. Active communication with broad stakeholders, such as government, consumers, academia, industry, NGO, media and others, will still be very critical for acceptance in China. 6. Suggestions and perspectives

opinions concerning risk and risk-related factors

Fig. 4. Components of risk analysis.

frequently. The Chinese government has emphasized risk-benefit communication with the public in terms of biotechnology since 2009/2010. No.1 Document issued in 2015 required increased public awareness of biotechnology. The MOA has conducted a promotion campaign, launched an official social media account to release related information specifically about biotechnology, and conducted training of media reporters to deliver accurate messages on biotechnology. In addition, the Chinese government has disclosed all approved safety certifications for biotech traits in China (import and cultivation) and implemented a public comment period once the regulations were under revision. However, risk communication on GM food safety in China is not yet well-established. A huge debate and concerns about GM food safety remain. In 2009, China issued biosafety certificates for commercial planting for two GM Bt rice lines and one GM phytase corn line, but no large-scale cultivation has occurred (Li, Peng, Hallerman, & Wu, 2014). One of the reasons for this lack of cultivation is the debate and concern about GM food safety.

Technology innovation in agriculture can play a key role in addressing challenges in China such as feeding growing population with limited arable land, adapting to climate change and protecting natural resources. It is essential to ensure food security and safety, which are top priorities for China. Genome editing is an important new technology with huge potential to improve agricultural productivity in China. Although genome-editing technology is a challenge for current detection methods and food-labeling systems, and some genomeediting techniques are likely to cause off-target mutations other than the intended mutation (Araki & Ishii, 2015), these hurdles will be overcome as the technology rapidly advances. Furthermore, discussions around the globe on regulatory frameworks will aid the establishment of science-based regulations for genome-editing techniques, especially CRISPR/Cas 9-edited products. The current simplified regulatory process for genomeedited products in some countries is mainly focused on base deletions by NHEJ. Whether this process is applicable for transgene insertions involving HR mechanisms, which allow the introduction of exogenous sequences, remains unresolved. Finally, as a very promising technology, genome editing not only has powerful applications in crop improvement and animal health but has also been widely used in drug discovery, which is more closely related to human health and life. To increase general understanding and confidence about genome editing-derived products as well as public support, active communication among government, industry, consumer, media and other interested stakeholders is needed and very critical. With appropriate regulation of genome editing, the public will receive increased benefits from genome editing-derived products. Conflict of interest

5.5. Risk analysis for genome-editing derived products in China The authors declare that they have no conflict of interest. As mentioned previously, discussion on risk analysis for genome-editing derived products in China has been initiated in China since 2015. NBC is responsible for technical review of all applications requesting safety certificates for biotech-derived products. A working group within NBC, was established in 2016 to provide technical assistance on how to do risk analysis on new techniques including genome editing in China. Based on discussions, the need to conduct safety assessment and the scope of those assessments on genome editing derived products depends on the types of variations generated following caseby-case principle. It was recommended that the products developed using site-directed transgene integration or transgene editing will continue to be regulated under the existing regulatory frameworks for GMOs and follow the same safety assessment process as GMOs. The types of variations generated by genomeediting techniques that are similar to or indistinguishable from the types of variations introduced by traditional breeding or

References Aghaee, M. A., Olkowski, S. M., Shelomi, M., Klittich, D. S., Kwok, R., Maxwell, D. F., et al. (2015). Waiting on the gene revolution: Challenges for adopting GM crops in the developing world. Trends in Food Science & Technology, 46(1), 132e136. Araki, M., & Ishii, T. (2015). Towards social acceptance of plant breeding by genome editing. Trends in Plant Science, 20(3), 145e149. Berman, J. R., Cooper, S., Zhang, B., Karlin-Neumann, G., Litterst, C., Jouvenot, Y., et al. (2015). Ultra-sensitive quantification of genome editing events using droplet digital PCR. Bio-Rad Laboratories, Inc. Bulletin 6712. Camacho, A., Deynze, A. V., Chi-Ham, C., & Bennett, A. B. (2014). Genetically engineered crops that fly under the US regulagory radar. Nature Biotechnology, 32(11), 1087e1091. Carlson, D. F., Lancto, C. A., Zang, B., Kim, E. S., Walton, M., Oldeschulte, D., et al. (2016). Production of hornless dairy cattle from genome-edited cell lines. Nature Biotechnology, 34(5), 479e481. Cicia, G., Caracciolo, F., Cembalo, L., Teresa, D. G., Grunert, K. G., Krystallis, A., et al. (2016). Food safety concerns in urban China: Consumer preferences for pig process attributes. Food Control, 60(2), 166e173. Cox, D. B. T., Platt, R. J., & Zhang, F. (2015). Therapeutic genome editing: Prospects

W. Gao et al. / Food Control 84 (2018) 128e137 and challenges. Nature Medicine, 21(2), 121e131. Cyranosk, D. (2016). Frist trial of CRISPR in people. Nature, 535(7), 476e477.
n ~ ez, M. S., & Alonso, D. B. (2016). An assessment of the barriers Entrena, M. R., Ordo to the consumers' uptake of genetically modified foods: A neural network analysis. Journal of the Scinece of Food and Agriculture, 96(5), 1548e1555. European Food Safety Authority (EFSA). (2012a). Scientific opinion addressing the safety assessment of plants developed through cisgenesis and intragenesis. EFSA Journal., 10(2), 2561. European Food Safety Authority (EFSA). (2012b). Scientific opinion addressing the safety assessment of plants developed using Zinc Finger Nuclease 3 and other Site-Directed Nucleases with similar function. EFSA Journal., 10(10), 2943. Fan, D., Liu, T. T., Li, C. F., Jiao, B., Li, S., Hou, Y. S., et al. (2015). .Efficient CRISPR/Cas9mediated targeted mutagenesis in Populus in the first generation. Scientific Reports, 5, 1e5. Federal Register. (2017). Genome editing in new plant varieties used for foods; request for comments. Food and Drug Adiminstration. Retrieved 04.24.17 https://www. federalregister.gov/documents/2017/01/19/2017-00840/genome-editing-innew-plant-varieties-used-for-foods-request-for-comments. Feng, Z., Mao, Y., Xu, N., Zhang, B., Wei, P., Yang, D., et al. (2014). Multigeneration analysis reveals the inheritance, specificty, and patterns of CRISPR/Cas-induced gene modifications in. Arbidopsis. Proc Natl Acad Sci, 111(12), 4632e4637. Feng, Z. Y., Zhang, B. T., Ding, W. N., Liu, X. D., Yang, D. L., Wei, P. L., et al. (2013). Efficient genome editing in plants using a CRISPR/Cas system. Cell Research, 23(10), 1229e1232. Food and Agriculture Organization (FAO). (2008). GM food safety assessment. Food quality and standards service, nutrition and consumer protection division. Rome: FAO of the UN. Fu, Y., Sander, J. D., Reyon, D., Cascio, V. M., & Joung, J. K. (2014). Improving CRISPR/ Cas nucleases specificity using truncated guide RNAs. Nature Biotechnology, 32, 279e284. Gaj, T., Gersbach, C. A., & Barbas, C. F., III (2013). ZFN, TALEN. and CRISPR/Cas-based methods for genome engineering. Trends in Biotechnology, 31(7), 397e405. Gao, J., Wang, G., Ma, S., Xie, X., Wu, X., Zhang, X., et al. (2015). CRISPR/Cas9-mediated targeted mutagenesis in Nicotiana tabacum. Plant Molecular Biology, 87(1), 99e110. Haun, W., Coffman, A., Clasen, B. M., Demorest, Z. L., Lowy, A., Ray, E., et al. (2014). Improved soybean oil quality by targeted mutagenesis of the fatty acid desaturase 2gene family. Plant Biotechnology Journal, 12(7), 934e940. Host-Jensen, A., Bertheau, Y., Loose, M. D., Grohmann, L., Hamels, S., Hougs, L., et al. (2012). Detecting un-authorized genetically modified organisms (GMOs) and derived materials. Biotechnology Advances, 30(6), 1318e1335. ILSI Focal Point in China. (2017). Workshop on development and challenge for agricultural biotechnology. Retrieved 07.14.17 http://www.ilsichina.org/foreend/ page.aspx?id¼31. ISAAA. (2016). GlobalStatusofCommercializedBiotech/GMCrops:2016. ISAAABriefNo.52. Ithaca,NY: ISAAA. Jia, H. G., & Wang, N. (2014). Targeted genome editing of sweet orange using Cas9/ sgRNA. PLoS One, 9(4), e93806. Ji, X., Zhang, H. W., Zhang, Y., Wang, Y. P., & Gao, C. X. (2015). Establishing a CRISPRCas-like immune system conferring DNA virus resistance in plants. Nature Plants, 1(10), 1e4. Jones, H. D. (2015). Regulatory uncertainty over genome editing. Nature Plants, 1(1), 1e3. Ledford, H. (2013). US regulation misses some GM crops. Nature, 500(8), 389e390. Liang, Z., Zhang, K., Chen, K. L., & Gao, C. X. (2014). Targeted mutagenesis in Zea mays using TALENs and the CRISPR/Cas System. Journal of Genetics and Genomics, 41(2), 63e68. Li, T., Liu, B., Spalding, M. H., Weeks, D. P., & Yang, B. (2012). High-efficiency TALENbased gene editing produces disease-resistant rice. Nature Biotechnology, 30(5), 390e392. Li, Y. H., Peng, Y. F., Hallerman, E. M., & Wu, K. M. (2014). Biosafety management and commercial use of genetically modified crops in China. Plant Cell Reports, 33(4), 565e573. Luo, Y. B. (2016). Risk-assessment of biotech derived food safety. Science Press, Beijing, 180. Lusser, M., Parisi, C., Plan, D., & Rodriguez-Cerezo, E. (2011). New plant breeding techniques (pp. 63e72). state-of-the-art and prospects for commercial development. EUR 24760, Spain. Mao, Y. F., Zhang, Hu, Xu, N. F., Zhang, B. T., Gou, F., & Zhu, J. K. (2013). Application of the CRISPR-cas system for efficient genome engineering in plants. Molecular Plant, 6(6), 2008e2011. Marchant, G. E., & Cardineau, G. A. (2013). The labeling debate in the United States. GM Crops and Food: Biotechnolgy in Agriculture and Food Chain, 4(3), 1e9.
noret, S., Fontanie re, S., Jantz, D., Tesson, L., Thinard, R., Re
my, S., et al. (2013). Me Generation of Rag1-knockout immunodeficient rats and mice using engineered meganucleases. The FASEB Journal, 27(2), 703e711. Miao, J., Guo, D. S., Zhang, J. Z., Huang, Q. P., Qin, G. J., Zhang, X., et al. (2013). Targeted mutagenesis in rice using CRISPR-Cas system. Cell Research, 23(10), 1233e1236. Ministry of Agriculture (MOA). (2002). Measures on labeling administration of Ag GMOs. Retrieved 04.24.17 http://www.moa.gov.cn/ztzl/zjyqwgz/zcfg/201007/ t20100717_1601302.htm.

137

Ministry of Agriculture (MOA). (2016). The 5th term of National Biosafety Committee was established. Retrieved 04.24.17 http://www.moa.gov.cn/zwllm/zwdt/ 201609/t20160908_5269516.htm. Ministry of Agriculture (MOA). (2017). Guideline for safety assessment of genetically modified organisms (GMOs). Retrieved 04.24.17 http://www.moa.gov.cn/zwllm/ zcfg/nybgz/201701/t20170124_5464913.htm. Miyaoka, Y., Chan, A. H., & Conklin, B. R. (2016). Detecting single-nucleotide substitutions induced by genome editing. Cold Spring Harbor Protocols, 672e675. Paul, J. W., III, & Qi, Y. (2016). CRISPR/Cas9 for plant genome editing: Accomplishments, problems and prospects. Plant Cell Rep, 35, 1417e1427. Perkel, J. (2013). Genome editing with CRISPRs, TALENs and ZFNs. Retrieved 04.24.17 http://www.biocompare.com/Editorial-Articles/144186-Genome-Editing-withCRISPRs-TALENs-and-ZFNs. Querci, M., Jermini, M., & Eede, G. V. D. (2006). The Analysis of food samples for the presence of genetically modified organisms. Joint Research Center, European Commission. Samanta, M. K., Dey, A., & Gayen, S. (2016). CRISPR/Cas9: An advanced tool for editing plant genomes. Transgenic Res, 25, 561e573. Schuttelaar, & Partners. (2015). The regulatory status of new breeding techniques in countries outside the European union. The Hague (NL). Shan, Q. W., & Gao, C. X. (2015). Research progress of genome editing and derivative technologies in plants. Hereditas (Beijing), 37(10), 953e973. Shan, Q. W., Wang, Y. P., Li, J., & Gao, C. X. (2014). Genome editing in rice and wheat using the CRISPR/Cas system. Nature Protocols, 9(10), 2395e2410. Shan, Q. W., Wang, Y. P., Li, J., Zhang, Y., Chen, K. L., Liang, Z., et al. (2013). Targeted genome modification of crop plants using a CRISPR-Cas system. Nature Biotechnology, 31(8), 686e688. Smith, J., Grizot, S., Arnould, S., Duclert, A., Epinat, J., Chames, P., et al. (2006). A combinatorial approach to create artificial homing endonucleases cleaving chosen sequences. Nucleic Acids Research, 34, e149. Sprink, T., Eriksson, D., Schiemann, J., & Hartung, F. (2016). Regulatory hurdles for genome editing: Process-vs. Product-based approaches in different regulatory contexts. Plant Cell Reports, 35(5), 1493e1506. The National People’s Congress of China (NPC). (2015). Food safety Law. Retrieved 04.24.17 http://www.npc.gov.cn/npc/zfjc/zfjcelys/2015-04/25/content_ 1992458.htm. The State Council of China (State Council). (2001). Regulation on administration of agricultural genetically modified organisms safety. Retrieved 04.24.17 http:// www.gov.cn/gongbao/content/2001/contentd_60893.htm. United States Department of Agriculture (USDA). (2017b). National bioengineered food disclosure standard. Retrieved 04.24.17 https://www.ams.usda.gov/sites/ default/files/media/Final%20Bill%20S764%20GMO%20Discosure.pdf. United States Department of Agriculture (USDA). (2017a). Questions & answers: APHIS requests public input on next step towards revision of its biotechnology regulations. Retrieved 04.24.17 https://www.aphis.usda.gov/biotechnology/ downloads/340/q&a_biotech-reg-revisions.pdf. Vilijoen, C. D., & Marx, G. M. (2013). .The implications for mandatory GM labeling under the Consumer Protection Act in South Africa. Food Control, 31, 387e391. Voytas, D. F., & Gao, C. X. (2014). Precision genome engineering and Agriculture: Opportunities and regulatory challenges. PLoS Biology, 12(6), 1e6. Waltz, E. (2016a). CRISPR-edited crops free to enter market, skip regulation. Nature Biotechnology, 34(6), 582. Waltz, E. (2016b). Gene-edited CRISPR mushroom escapes US regulation. Nature, 532(4), 293. Wang, Y. P., Cheng, X., Shan, Q. W., Zhang, Y., Liu, J. X., Gao, C. X., et al. (2014). Simultaneous editing of three homoeoalleles in hexaploid bread wheat confers heritable resistance to powdery mildew. Nature Biotechnology, 32(9), 947e951. Whelan, A. I., & Lema, M. A. (2015). Regulatory framework for gene editing and other new breeding techniques (NBTs) in Argentina. GM Crops and Food Biotechnology in Agriculture and the Food Chain, 6(4), 253e265. Wolt, J. D., Wang, K., & Yang, B. (2016). The regulatory status of genome-edited crops. Plant Biotechnology Journal, 14, 510e518. World Health Organization (WHO), Food and Agriculture Organization (FAO). (2016). Risk communication applied to food safety handbook. Rome: WHO/FAO. Zhang, Y., Liang, Z., Zong, Y., Wang, W. P., Liu, J. X., Chen, K. L., et al. (2016). Efficient and transgene-free genome editing in wheat through transient expression of CRISPR/Cas9 DNA or RNA. Natural Communications, 8, 1e8. Zhang, H., Zhang, J., Wei, P., Zhang, B., Guo, F., Feng, Z., et al. (2014). The CRISPR/Cas9 system produces specific and homozygous targeted gene editing in rice in one generation. Plant Biotechnology Journal, 12(6), 797e807. Zhao, L., Gu, H., Yue, C., & Ahlstrom, D. (2013). Consumer welfare and GM food labeling: A simulation using an adjusted kumaraswamy distribution. Food Policy, 42, 58e70. Zhou, Q. (2016). Genome editing in China. Guangming Daily, 2016-08-12. Zhuo, Q. (2014). Analysis of the worldwide management for labeling of genetically modified food. Journal of Chinese Institute of Food Science and Technology, 14(8), 16e20. Zong, Y., Wang, Y. P., Li, Ch, Zhang, R., Chen, K. L., Ran, Y. D., et al. (2017). Precise base editing in rice, wheat and maize with a Cas9-cytidine deaminase fusion. Nature Biotechnology, 35(2), 1e3.