Genetically Engineered Crops Experiences and Prospects (2016) / Chapter Skim
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7 Future Genetic-Engineering Technologies
7 Future Genetic-Engineering Technologies
Pages 353-404
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approaches are reviewed to evaluate their potential to assess intended and unintended effects of genetic engineering and conventional plant breeding. The committee concludes that advances in genetic engineering and -omics technologies have great potential to enhance crop improvement in the 21st century, especially when coupled with advanced conventional-breeding methods.
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MAS and genomics greatly reduce the number of individual plants that need to be retained in the breeding pipeline for phenotyping. Genome-level datasets and genomic technologies have been used to identify causal genes, alleles, and loci important to relevant agronomic traits and have thereby become tools to accelerate breeding cycles (Box 7-1)
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. Access to the potato genome sequence permitted identification of the allelic variants of the gene that controls tuber initiation, which not only provided a clear connection between tuberiza tion, the circadian clock, and the tuberization signal but provided a molecular marker for use in MAS to identify varieties adapted to specific geographic regions (Kloosterman et al., 2013)
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. At the time when the committee's report was being written, a reference genome of nearly every major crop species was available.
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This type of "one-gene" genetic engineering was exemplified in most commercialized crops grown in 2015. Transgenics versus Cisgenics versus Intragenics Because of legislative, regulatory, marketing, and public-perception concerns, efforts have been made to develop GE crops with genes found in a crop species of interest or a plant species that can naturally interbreed with it (Rommens, 2004)
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cells. Nonetheless, there is tremendous 2 See Chapter 3 (section "The Development of Genetic Engineering in Agriculture")
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It had been known since the earliest days of plant biotechnology that post-transcriptional gene regulation (silencing) was an important process that could regulate the level of expression of plant genes.
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As with any genetic engineering-based insect-control strategy, potential nontarget effects need to be investigated. Development of Non–Tissue-Culture Transformation Methods As described in Chapter 3, the construction of GE plants commonly relies on in vitro plant tissue culture, transformation, and plant regeneration.
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The technologies include genome editing, synthetic DNA components and artificial chromosomes, and targeted epigenetic modifications.
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Four main classes of SSNs are used in plant genome editing (reviewed in Voytas and Gao, 2014) : meganucleases, zinc finger nucleases (ZFNs)
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Their ease of design for specific target DNA sequences revolutionized genome editing. In nature, Xanthomonas species secrete TALEs into plant cells to enable pathogenicity.
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, and Clustered regularly interspaced palindromic repeats (CRISPR)
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At the time the committee's report was being written, the CRISPR/Cas system used in genome editing was primarily the Type II CRISPR/Cas9, from Streptococcus pyogenes, in which foreign DNA sequences are incorporated between repeat sequences at the CRISPR locus and then transcribed into an RNA molecule known as crRNA (reviewed by Sander and Joung, 2014)
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Genome editing via CRISPR and other techniques might be performed in plants via transient expression of transgenes (Clasen et al., 2016) or without exogenous DNA at all (Woo et al., 2015)
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DNA constructs used in plant genetic engineering have been small (less than 20–40 kilobases) and have used traditional molecular cloning techniques that are slow and laborious.
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can be fused to a transcriptional activator (green) and, in the presence of a guide RNA (orange and yellow)
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and, in the presence of a guide RNA (orange and yellow) , guide the complex to the promoter of a gene of interest and decrease transcription of the target gene.
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annua, this proof-of-concept study demonstrated the feasibility of engineering heterologous biosynthetic pathways in plants. With further refinement of promoters, improved understanding of metabolite compartmentalization and transport and flux in cells and tissues, and development of such vectors as artificial chromosomes capable of transferring large segments of DNA to plant cells, genetic engineering of plants for complex traits, such as heterologous or novel biochemical pathways, and for specialized physiological
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From page 371... ...
Thus, the safety issue is the same as that involved in any technique, whether genetic engineering or non-genetic engineering, that results in an increase or decrease in the expression of specific genes in a plant and in specific trait alterations. It should be noted that, in addition to targeted approaches, there are several ways to broadly and randomly alter the epigenome of a plant, such as overexpression of an enzyme that alters DNA methylation.
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In this section, the committee provides an overview of some of the expected applications of this transformation technology. Removal of Genome-Editing Reagents in Genetically Engineered Crops It is envisioned that genome editing will be useful in most agricultural crops in generating modified alleles that are homozygous in the modified line for several reasons: to prevent segregation of the altered allele in derived progeny, to eliminate the production of the wild-type target mRNA and protein, and to increase the dosage of the modified allele as the level of transcript of a gene is correlated with numbers of alleles.
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Another way is to employ DNAfree or transgene-free genome editing (Woo et al., 2015) in which only proteins and RNAs are introduced into the plant to accomplish gene editing.
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To demon strate whether TALEN-mediated cleavage of the MLO loci could be used to edit the locus, a donor molecule encoding either the green fluorescent protein or a histidine-tagged protein was c otransformed along with the TALEN, resulting in integration of the donor molecule into the MLO locus. This type of modification is referred to as a "knock-in." Preliminary work with a single CRISPR guide RNA tar geted to TaMLO-A1 yielded plants with mutations at this locus; this indicated the feasibility of achieving disease resistance in wheat with two distinct site-specific nuclease genome-editing technologies.
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. NOTE: A, the polyploid wheat genome encodes three dominant MLO genes that confer susceptibility to the fungal powdery mildew pathogen.
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The chemical mutagenesis used in TILLING introduces random mutations into the plant genome; although most of the mutations can be removed by backcrossing in most crop species, the resulting modi fied crop plant might have more unknown changes than the same change in the target gene of interest brought about by use of CRISPR/Cas 9 (al though somaclonal variation will not be an issue because TILLING does not require a tissue-culture step)
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Multiple backcrosses must be made to remove unlinked introgression events, which is expensive in terms of growing populations in the greenhouse or field for multiple generations. Thus, genome editing of QTL provides an alternative approach for developing elite varieties in species whose breeding cycles pose logistical challenges.
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EMERGING TECHNOLOGIES TO ASSESS GENOME-EDITING SPECIFICITY A highly touted feature of emerging genome-editing methods is their extreme specificity -- ZFNs, TALENs, and the CRISPR/Cas9 nuclease system rely on recognition of a target sequence, so a single nucleotide in a genome can be targeted and modified. However, the extent of off-target effects is not well established, and off-target effects would potentially have unintended effects.
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What was most notable about the Tsai et al. study was that a substantial reduction in off-target effects was observed by using truncated guide RNAs; this suggests that improvements in the design of the CRISPR reagents have strong potential to affect the specificity of genome editing.
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An analogous project that uses diverse -omics approaches in crop plants with genetic engineering and conventional breeding could provide in-depth improvements in the understanding of plant biological processes that in turn could be applied to assessing the e ffects of genetic modifications in crop plants. Genomics One way to ascertain whether genetic engineering has resulted in offtarget effects (whether through nuclear transformation with Agrobacterium or gene guns, RNAi, or such emerging technologies as genome editing)
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thaliana reference genome sequence and with the availability of sequences from more than 800 additional accessions,8 an estimated 30–40 million nucleotides of sequence were still missing from the A thaliana Col-0 reference genome assembly (Bennett et al., 2003)
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. Resequencing: Assessing Differences Between the Reference and Query Genome Once the DNA sequence of a crop's genome is assembled well enough to serve as a reference genome, resequencing becomes a powerful and costeffective method for detecting genomic differences among related accessions (individuals)
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A, genome sequencing is performed on both the wild-type and genome-edited accession, and differences in the DNA sequence (red G) are detected with bioinformatics methods.
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Fifth, read alignments and polymorphism detection are limited to nonrepetitive regions of the genome, so regions that are repetitive in the genome cannot be assessed for divergence. Although obstacles remain, resequencing is a powerful method for measuring differences in genome sequences between wild-type plants (normal untransformed individuals)
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Utility of Transcriptomics, Proteomics, and Metabolomics in Assessing Biological Effects of Genetic Engineering As stated in the 2004 National Research Council report Safety of enetically Engineered Foods, understanding the composition of food at G the RNA, protein, and metabolite levels is critical for determining whether genetic engineering results in a difference in substantial equivalence compared to RNA, protein, and metabolite levels in conventionally bred crops (NRC, 2004; see Chapter 5)
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, total RNA, RNA bound to ribosomes, and RNA-protein complexes to gain a detailed assessment of RNAs in a cell. Methods to construct RNA-seq libraries, generate sequence reads, align to a reference genome, and determine expression abundances are fairly robust even with draft genome sequences if they provide nearly complete representation of the genes in the genome (Wang et al., 2009; de Klerk et al., 2014)
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It is impossible to determine whether the changes in transcript levels recorded in the study indicate that the GE rice might be worse than, equal to, or better than its non-GE counterpart as regards food safety. One way to assess the biological effects of genetic engineering on the transcriptome is to include a variety of conventionally bred cultivars in the study and determine whether the range of expression levels in the GE line falls within the range observed for the crop, but this method will not provide definitive evidence of food or ecosystem safety.
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The mass spectra are compared with a standard library of chemicals run on the same analytical system. The major problem for this type of metabolomic analysis of plants is the possession in the plant kingdom of large numbers of genus-specific or even species-specific natural products (see section "Comparing Genetically Engineered Crops and Their Counterparts" in Chapter 5 for discussion of plant natural products)
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Generally, with a few exceptions, metabolomic studies have concluded that the metabolomes of crop plants are affected more by environment than by genetics and that modification of plants with genetic engineering typically does not bring about off-target changes in the metabolome that would fall outside natural variation in the species. Baseline studies of the metabolomes (representing 156 metabolites in grain and 185 metabolites in forage)
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Epigenetic marks are determinants of transcriptional competence, and alteration of the epigenetic state (which occurs naturally but infrequently) can alter expression profiles or patterns of target genes.
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As a consequence, nonmethylated cytosines will be detected as thymidines after the polymerase chain reaction step during epigenome-library construction. After sequencing, reads are aligned with a reference genome sequence, and nonmethylated cytosines are detected as SNPs and compared with a parallel library constructed from untreated DNA (see section above "Resequencing: Assessing Differences Between the Reference and Query Genome"; Figure 7-5)
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Also, the present state of understanding does not permit robust prediction of the effects of many epigenetic modifications on gene expression, and gene expression can be more thoroughly and readily assessed by transcriptomics. Evaluation of Crop Plants Using -Omics Technologies The -omics evaluation methods described above hold great promise for assessment of new crop varieties, both GE and non-GE.
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In Tier 1, there are no differences between the variety under consideration and a set of conventionally bred varieties that represent the range of genetic and phenotypic diversity in the species. In Tier 2, differences are detected that are well understood to have no expected adverse health or environmental effects.
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It is also straightforward and relatively low in cost to generate genomesequence data from many individuals from a new GE or non-GE variety to determine which lineage has the fewest nontarget changes to its genome. As noted earlier in the chapter, mutagenesis, although currently classified as conventional breeding, can result in extensive changes to the genome; thus generating DNA sequence data will be useful in evaluating varieties produced by this method.
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should be constructed for the range of variation inherent in both con ventionally bred and genetically engineered crop species. CONCLUSIONS Modern plant breeding and genetic engineering are complementary methods for improving crop yield, production efficiency, and composition.
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It is important that basic knowledge regarding biology continues to grow and that technology continues to improve. In addition to contributing to crop improvement, emerging -omics methods could provide a rational pathway to developing a tiered approach for assessing health and environmental effects of new crop varieties produced by conventional breeding and genetic engineering.
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2014. Genome editing.
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2011. Determining DNA methylation profiles using sequencing.
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Nature Genetics 44:808–811. International Barley Genome Sequencing.
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2011. Covering chemical diversity of genetically-modified tomatoes using metabolomics for objective substantial equivalence assessment.
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2014. Comprehensive characterization of complex structural variations in cancer by directly comparing genome sequence reads.
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Plant Physiology 131:866–871. Potato Genome Sequencing Consortium.
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2010. Genome editing with engineered zinc finger nucleases.
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2015. DNA-free genome editing in plants with preassembled CRISPR-Cas9 ribonucleoproteins.
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