How to administer genetically edited crops in China

[HC360] On the 28th of last month, US Agriculture Minister Sonny Perdue said in a statement that some genetically edited crops would not be regulated. Since 2016, the US Department of Agriculture has released some genetically edited crops. This time, it is clear that it has confirmed its position.

For genetic engineering, including the regulation of genetically modified organisms, the United States generally targets products rather than technology. This strategy guarantees a global leadership in agriculture. At present, the United States will continue to lead the world today when genetic editing technology revolutionizes crop breeding; in contrast, Europe and China are already lagging behind.

(1) It is impossible to become possible

In July 2014, a collaboration between Gao Caixia of the Institute of Genetics of the Chinese Academy of Sciences and the Qiu Jinlong research group of the Institute of Microbiology was published on Nature Biotechnology. They created a new wheat variety with broad-spectrum and long-lasting resistance to powdery mildew. It's also simple, they just deleted a few of the 17,000,000,000 bases of the wheat genome. This was impossible in the past.

In Europe, broad-spectrum anti-white powder barley varieties have been widely used in production. The broad-spectrum resistance of barley to powdery mildew is due to the functional deletion mutation of the MLO gene. This mutation, originally found in wild barley in Africa, was later discovered by breeders and introduced into barley grown in Europe by means of hybridization. And since then, although after more than 30 years of production and application, this resistance is still effective. The problem is that although barley and wheat are closely related, they have reproductive isolation and cannot be crossed.

How to make wheat also have broad-spectrum resistance to powdery mildew?

In 2010, they worked together to solve this problem with the new gene editing technology TALEN. "Wheat is hexaploid (AABBDD), and there is a copy of the MLO gene in the common wheat A, B, and D genomes. We used the genome editing technique to simultaneously mutate these three copies and obtained a wide range of powdery mildew. Spectrum-resistant wheat.” Qiu Jinlong told Intellectuals.

Through this experiment, they also understand why wheat does not acquire the same broad-spectrum resistance as barley through natural mutations. In the experiment, they obtained 35 mutants of the MLO gene, with single-copy, double-copy, and three-copy mutations. They obtained self-intersection of these mutants and obtained homozygous mutant progeny (aa, bb, dd, aabb, aadd, bbdd, aabbdd) of different combinations of MLO genes, only to find that only aabbdd, that is, MLO gene in wheat A Plants with simultaneous mutations in the three copies of the B and D genomes have broad-spectrum resistance.

“Barley is diploid, wheat is hexaploid, there are three copies of each genome, and there are six alleles. In nature, the probability of simultaneous mutation of six alleles is almost zero, which is why it does not exist. Naturally mutated MLO broad-spectrum wheat against powdery mildew," Qiu Jinlong said.

(B) natural mutations

It is possible to make precise changes to DNA sequences, and genetic fixed-point editing technology is setting off an agricultural revolution. Creating wheat that is resistant to powdery mildew is just a small test.

Gene editing techniques benefit from the design and use of several nucleases. A common feature of these nucleases is that they can produce double-strand breaks at the DNA site you want, which can be understood as "what to call". Subsequently, the intrinsic repair mechanism within the cell is activated, producing efficient genetic modifications to the lesion. Several currently used nucleases include zinc finger nucleases (ZENs), transcription-activator-like effector nucleases (TALENs) and RNA-directed endonucleases based on the CRISPR/Cas system, all of which are specific for DNA. The site is cut.

There are two ways to repair DNA double-strand breaks in cells: one is called non-homologous end joining, referred to as NHEJ; the other is called homologous recombination, referred to as HR. In contrast, NHEJ is the primary repair mechanism, usually resulting in the loss of a small number of bases, and in some cases base insertions.

Because NHEJ is relatively simple to implement, it mostly leads to gene knockout, making it a good tool for studying gene function. After understanding the function of the gene and predetermining how to modify the DNA sequence, you can use the gene editing technology to generate mutations accurately and quickly.

Gene knockouts make their loss function seem useless, but in fact some plants produce toxic substances or allergens that need to be removed before consumption. In addition, the plant itself will also produce some substances that affect production, storage and processing, and the elimination will bring benefits. For example, in 2016, plant pathologist Yang Yinong of Penn State University in the United States used gene editing technology to knock out one of the six genes encoding polyphenol oxidase (PPO) in mushrooms, creating an anti-browning mushroom. Storage and transportation.

Even more surprising is that knocking out certain genes may also increase nutrients. For example, the loss of certain fatty acid desaturase in seeds can accumulate monounsaturated fat, so that the oil extracted from the mutant seeds is healthier and the shelf life is also improved. Longer.

The result of such a mutant is that only a few bases have been deleted, and it has been indistinguishable from natural mutations, and mutants produced by artificial mutagenesis such as chemical reagents and X-rays. "This also brings difficulties to the test, because it is difficult to distinguish between the two." Tsinghua University professor Xie Zhen told the "Intellectuals".

In 2015, a British plant molecular geneticist Huw D. Jones pointed out in a commentary by Nature Plants that apart from documenting new traits, it is impossible to make a clear distinction, so if global trade is considered, the EU will In a dilemma of regulation: “If you import products from countries that have genetically edited crops, do you want to close one eye to an unapproved product that may be shipped, and do not treat these products as genetically modified crops. Or you have to ban all imports of commercial crops from these countries, whether or not they are genetically engineered."

In fact, for the above-mentioned gene knockout mushroom, corn, the US Department of Agriculture has indicated that it is not within its scope of supervision.

"We call them natural mutations." Qiu Jinlong said. In fact, not only the final product is similar to conventional breeding, but also the use of gene editing technology is more accurate, simpler and more direct; while traditional hybridization often leads to the exchange of large fragments of the genome, even the mutation breeding leads to thousands of random mutations. It requires a large number of offspring screening, which is time consuming and laborious.

(3) Without any genetic modification

Of course, if you study the creation process of broad-spectrum wheat powder-resistant wheat, it is not “invulnerable”. The main problem is that the nuclease is a protein. If the DNA encoding the protein, or the plasmid carrying the DNA, is introduced into the plant cell and randomly integrated into the chromosome of the plant, it is also the insertion of foreign DNA.

One solution is to further screen for the removal of plants containing recombinant DNA after nuclease expression and plant production. The ability to do this is due to the fact that the two sites originally designed are not tied together. These two sites, one is the site of the DNA encoding the nuclease, and the other is the site of action of the nuclease. These two sites are far apart, even if the nuclease-encoding DNA is integrated into the chromosome of the plant, and then can be separated by backcrossing, leaving the plant progeny carrying only the desired DNA sequence changes.

However, the process of hybrid screening is time consuming and labor intensive. Moreover, stable expression of the nuclease DNA increases the probability of off-target and chimerism. The specificity of off-target and nuclease refers to the fact that nucleases "carelessly" cut similar sites due to the similarity of certain DNA fragments.

Fortunately, the rapid development of gene editing technology can achieve the so-called "transient expression", overcome the above shortcomings, and completely eliminate the genetic modification.

In the first mode, the nucleic acid encoding DNA or RNA is transferred to plant cells using Agrobacterium, gene gun or protoplast transformation. Transient expression is often produced when a nuclease-encoding DNA construct is delivered to a cell. After expression of the nuclease, the DNA construct degrades rapidly and there is no opportunity to integrate into the plant genome.

As an example, in August 2016, Gao Caixia's research group used the transient expression of CRISPR/Cas9 DNA or RNA to perform a fixed-point knockout of seven different genes of hexaploid wheat and tetraploid wheat. The wheat homozygous knockout mutant of the exogenous gene did not detect the off-target effect. "This is the role of instant, it is equivalent to wielding a knife to cut people, cut the enemy, but also hurt friends, the longer the enemy is cut, the greater the probability of hurting friends. Therefore, the transient expression will reduce off-target. It also shows that the accuracy is higher.” Qiu Jinlong said.

However, after the CRISPR/Cas9 DNA enters the cell, it is also possible to integrate the degraded small DNA fragment into the genome of the plant. How to fundamentally eliminate DNA or RNA from entering the cell?

The upgrade method is to transport the nuclease directly to the plant cell in the form of protein. Currently, ribonucleoprotein complexes (composed of Cas9 protein and guide RNA) assembled in vitro are directly transported into maize embryos by gene guns, and maize containing no transgenes is produced. The Gao Caixia team also used a similar method to achieve fixed-point editing in hexaploid wheat. The resulting wheat not only had no exogenous DNA from the final product, but also had no exogenous DNA throughout the creation process.

Equally powerful is the fast-growing base editing technology that directly alters bases without introducing DNA. Gao Caixia said in a commentary published in Nature Reviews Molecular Cell Biology in February this year, although the current base editing can only convert C to T, A to G, and the sequence editing window is narrower, but these The restrictions may be overcome in the coming year.

"There are some epigenetic modification techniques. The DNA sequence does not change at all, but it also produces new traits that cannot be detected." Xie Zhen said. Of course, whether an epigenetic modification technique, such as RdDM, produces a plant that can be called a mutant is also questionable because the nucleotide sequence does not change.

In short, the development of gene editing technology has not only obtained plants without transgenics, but also does not involve transgenes throughout the process.

(4) Fundamental errors

However, if you are talking about another way to fix HR, the problem can get complicated. HR achieves precise gene editing through gene replacement or insertion, and it is inevitable to introduce a DNA repair template similar to the original sequence at the break. The template is copied to the chromosome by homologous recombination for repair purposes.

HR has great potential. Traditional transgenes, foreign genes are randomly integrated into the genome, and sites targeting transgene insertion are known in advance. In addition, multiple transgenes can be inserted into the same site (stack) at once. Not only is the addition of genes, HR can also acquire new traits by altering key amino acid residues in the coding region of the gene, or by altering the promoter or other cis element that controls gene expression. For example, in order to get the herbicide-tolerant properties of the crop, one of the amino acids of acetolactate synthase (ALS) can be replaced, and HR can do this.

The insertion and replacement of bases may trigger regulation. However, if compared with traditional cross-breeding, if the homologous genes in the hybridizable species already existing in nature are directed, there is no reason not to accept such crops - because, in principle, such crops Crossbreeding can also be done, but it is more time consuming. Even more tricky is that there is no way to distinguish between products created by these two methods. The only difference is that traditional hybridization is time-consuming, low-accuracy, and often introduces a large portion of DNA in the vicinity, which may have side effects.

Then, if the homologous gene of a hybridizable species is introduced through HR, and the exogenous DNA is not a non-hybridizable species, can an exemption be obtained? In addition, how many base changes will trigger regulation? These problems still Unresolved.

However, in the opinion of some experts, considering whether the technology is involved in the process of genetic modification, such an idea is inherently wrong. In 2016, Gregory Conko of the American Institute of Competitive Enterprises and three other scholars reiterated that the “Stanford Model” proposed in 1997 not only continues to apply, but also does not depend on technological changes. They point out: "The bigger shortcoming of existing process-based regulations is that they always lag behind the introduction of new technologies. One example is the development of CRISPR-Cas9 and other gene editing technologies that have caused debate on how to conduct regulation. The debate that is completely invisible to the North is often focused on exploring which hypothetical category the crops to which these techniques are applied should be classified, such as whether it is genetically modified, whether it is a regulatory category, or whether it is a plant containing insect-resistant substances. .

Gregory Conko and others emphasize that regulation must focus on the actual risks that are only brought about by the modified end product and have nothing to do with the method used. The assessment of the final product, rather than the process of creating the product, represents the mainstream opinion of the scientific community.

(5) Miserable stories

In 2009, when Qiu Jinlong returned to China from Europe and was ready to use the emerging genetic editing technology TALEN to create wheat with broad-spectrum resistance to powdery mildew, his collaborator Gao Caixia also returned from Europe this year. I have arrived in China. Previously, she was a research scientist and research team leader in the research department of DLF-Trifolium in Denmark.

The conservative nature of genetic modification in Europe has led to the backwardness of its product development and industry. Therefore, when genetic editing and other new breeding technologies emerged, European scientists were keen to explore to avoid regulation and win public support. Their ultimate goal is to shift from process regulation to product regulation in Europe, but in reality it can only be hoped that some of the fixed-point genetic editing products will not be included in the regulation of genetic modification.

As early as 2011, the EU had a series of regulatory discussions on new breeding technologies, and an international symposium was held to make several classifications of fixed-point genome editing techniques and give regulatory advice. However, the rapid response of the EU has not reversed the situation. In the past few years, even the research on gene editing technology has been affected, let alone the application.

“In Europe, when we introduced, researchers are not accustomed to using this new technology, which is relatively conservative in Europe. In China, our plant genome editing research is still leading in the world, and it has been fast and extensive. Application." Qiu Jinlong said. Data show that from 2007 to 2011, 35% of the scientific publications of genetic editing were from Europe, but were later surpassed by the United States. Some commented that this may be due to the United States' more open and tolerant environment for technological innovation.

Gregory Conko and others believe that the United States has a relatively loose attitude toward genetic engineering technology compared to the European Union. The US Food and Drug Administration has consistently evaluated the food and feed obtained from recombinant DNA technology with the products obtained from traditional breeding techniques, and evaluated its products in a concentrated manner. Considering the equivalence, toxicity, allergies, and resistance of its components through informal consultation. Nutrition and other aspects. The US Environmental Protection Agency focuses on insect resistance and focuses on the “plant insecticides” – the environmental impact of expressed insect-resistant proteins. As the framework-leading regulator, the US Department of Agriculture invented the term "Plant pest" to focus on the use of plant pathogens in the creation of genetically engineered crops. For the emerging plant editing technology, the White House now orders the Ministry of Agriculture, the Food and Drug Administration, and the Environmental Protection Agency to update their biotechnology regulatory coordination framework to respond to changes in new technologies, while at the same time widely collecting opinions.

Traditional breeding has been unable to meet the growing environmental challenges of food demand and climate change. Crop improvement will not be sustainable if it is still found through natural mutations and cross-breeding, even with mutation breeding developed 60 years ago. "Traditional crossbreeding, suppose that it is fortunate to find a disease-resistant plant, but generally its yield is not high, then it needs to be hybridized with high yield, assuming hybridization, but also through multiple generations of optimized separation, generally at least ten More than a year." Qiu Jinlong said.

As a supplement to traditional technology, the genetically modified technology developed in the 1990s has created a large number of varieties of insect-resistant, herbicide-resistant, herbicide-rich, and has been cultivated for 23 years. According to data released by the International Agricultural Biotechnology Application Service (ISAAA), in 2016, 26 countries in the world have more than 180 million hectares of GM crops, while in the United States, more than 90% of soybeans and corn have one or more genetically modified organisms. So that they are resistant to insects or herbicides.

However, a European scientist, Maria Lusser et al., published an article in Nature Biotechnology in 2012, stating that the unfavorable regulatory environment resulted in high costs ($35 million per genetic event) and time-consuming (required 5.5 years to complete), making only a few High-yielding crops are grown on a large scale, such as cotton, soybeans and corn; some unpopular crops such as vegetables and horticultural varieties are left unattended.

“I think that if the genetic editor is sufficiently loose and does not need a big company to do it, it is very conducive to the innovation of small companies in China. We have written materials for the Academy of Sciences and used a very common term to say that we can achieve the curve of the Chinese breeding industry. Overtaking; China has more than 2,000 seed companies, mostly small companies, unable to compete with foreign multinational companies." Qiu Jinlong told the "Intellectuals."

The establishment of a science-oriented, product-based management system has been placed in front of governments. In the field of plant breeding, we are standing at the crossroads of change, perhaps a dual paradigm shift: on the one hand, plant editing technology with great potential is revolutionizing the industry; on the other hand, process-based regulatory strategies have Out of date.

How will China respond to the GM? China’s intellectuals know that information is scarce and vague – we can only hear “suggestions” from a few academic conferences, even scientists in this field for various reasons. , has been "cautious", and is not as deep as it is. The broad-spectrum wheat powder-resistant wheat that was born in Chinese laboratories is growing in the experimental fields of the United States because it does not need to be regulated by genetically modified organisms.

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Editor in charge: Chen Wei