One of the major limitations of the first-generation rDNA-based GM methods is the randomness of DNA insertions into plant genomes, just as the earlier mutagenesis methods introduced mutations randomly. The newer methods increase the specificity and precision with which genetic changes can be made. Known under the general rubric of sequence-specific nuclease (SSN) technology or gene/genome-editing, this approach uses proteins or protein-nucleic acid complexes that bind to and cut specific DNA sequences.1 SSNs include transcription activator-like effector nucleases (TALENs), zinc-finger nucleases (ZFNs), and meganucleases.2
[This is part three of a four-part series on the progress of agricultural biotechnology. Read part one and part two.]
The DNA cuts made by SSNs are repaired by cellular processes that often either change one to several base pairs or introduce deletions and/or insertions (aka indels) at the target site. Another recently added technology capable of editing gene sequences is termed oligonucleotide-directed mutagenesis (ODM) and uses short nucleic acid sequences to target mutations to selected sites.3
The hottest and the coolest
What is rapidly emerging as the most powerful of the SSN technologies is known by the uninformative acronym CRISPR/Cas, which contracts the unwieldy designation clustered regularly interspaced short palindromic repeats (CRISPR)CRISPR-associated protein (Cas9). Its based on a bacterial defense system against invading viruses and promises extraordinary versatility in the kinds of genome changes that it can make.1,4
The CRISPR/Cas editing molecular machine is comprised of an enzyme (Cas9 and other variants) that binds an RNA molecule (called the guide RNA or gRNA) whose sequence guides the complex to the matching genomic sequence, allowing the Cas9 enzyme to introduce a double-strand break within the matching sequence. The CRISPR/Cas system can be used to edit gene sequences, to introduce a gene or genes at a pre-identified site in the genome, and to edit multiple genes simultaneously, none of which could be done with rDNA methods.1,5
Many of the genetic changes created using either SSN or ODM are indistinguishable at the molecular level from those that occur in nature or are produced by mutation breeding. Since both spontaneous mutants and chemical- and radiation-induced mutants have been used in crop improvement without regulation, there is no scientific rationale for regulating mutants produced by the newer methods. In hopes of creating a distinction that will permit exemption of gene-edited crops from regulation, the newer methods are increasingly referred to as new plant breeding techniques (NPBTs or just NBTs).
Quick successes for NBTs?
Prime targets of gene editing are cellular proteins that are involved in pathogenesis.6 Virus reproduction requires the recruitment of cellular proteins for replication, transcription and translation. There can be sufficient redundancy in the requisite protein infrastructure so that partial or complete virus resistance can be achieved by disrupting genes that code for proteins required for viral replication without damaging crop productivity.
For example, work with mutants of the model plant Arabidopsis identified translation initiation factor eIF4E as required for potyvirus translation. CRISPR/Cas-induced point mutations and deletions have recently been reported to enhance viral resistance not only in Arabidopsis, but in cucumber and cassava, as well.7
The many ways that plants and their bacterial and fungal pathogens interact offer opportunities to use gene editing to enhance plant disease resistance and reduce agricultures dependence on chemical control agents.6 The two main strategies are to inactivate genes whose products render the host plant sensitive to pathogen invasion and to enhance the ability of the host plant to resist invasion by providing functional resistance factors they lack.
An example of the former is provided by the mildew resistance resulting from the inactivation of all three homeoalleles of the mildew resistance locus (MLO) of hexaploid wheat.8 The efficiency of targeting both multiple alleles and multiple loci has taken a further jump with the development of multiplexed gene editing using vectors carrying several gRNA sequences capable of being processed by cellular enzymes to release all of them. This allows the gRNAs to edit multiple genes simultaneously.9
The second approach is to capitalize on the formidable arsenal of resistance genes residing in plant genomes.10 Fungal resistance genes have long been a major target of breeders efforts and have proved frustratingly short-lived, as pathogens rapidly evolve to evade recognition.11 While desirable resistance genes missing from domesticated crops still reside in wild relatives, extracting them by conventional breeding methods can be time-consuming or impossible.
European academic researchers created transgenic potatoes resistant to the late blight (Phytophthora infestans) that caused the Irish potato famine by inserting resistance (R) genes cloned from wild potato species into commercial potato varieties.12 A blight-resistant variety, called the Innate Generation 2 potato, is being commercialized by J.R. Simplot company in the U.S. and Canada and is already being marketed in the U.S. as the White Russet Idaho potato.13 Transgenic disease-resistance traits have been introduced in other crops, but have yet to be commercialized.14
Plant genomes contain hundreds to thousands of potential R genes, but it is not yet possible to determine whether a given one will confer resistance to a particular pathogen. Methods are currently being developed to accelerate the identification and cloning of active ones.14 Once identified, CRISPR/Cas can be used to introduce cassettes carrying multiple R genes, making it possible to create more durable resistance than can be achieved by introducing a single R gene through conventional breeding14. Finally, direct editing of resident inactive R genes using a ribonucleoprotein (RNP) strategy that avoids creating a transgenic plant may prove useful, although no such products appear to be in the pipeline to commercialization at present.15,16
Multiplexed editing has proved particularly useful for editing genes in polyploid species. For example, Cas9/sgRNA-mediated knockouts of the six fatty acid desaturase 2 (FAD2) genes of allohexaploid Camelina sativa was reported to markedly improve the fatty acid composition of Camelina oil.17 Using a different approach, Yield10 Biosciences is moving toward commercialization of a high-oil Camelina developed by editing a negative regulator of acetyl-CoA carboxylase.18
As of this writing, the only gene-edited product that has been commercialized is a soybean oil with no trans-fat, trademarked Calyno, developed by Calyxt.19 Gene-edited crops that have been approved but not commercialized or are still in the regulatory pipeline include miniature tomatoes, high-fiber wheat, high-yield tomatoes, improved quality alfalfa, non-browning potatoes and mushrooms, as well as high starch-content and drought-resistant corn, most being developed by small biotech companies.19
Getting beyond the low-hanging fruit
It is becoming increasingly clear that yield increases in our major crops by traditional breeding approaches are not keeping pace with demand.20 The gap is likely to widen as climate warming moves global temperatures farther from those prevailing when our crops were domesticated.
Overexpression of stress-related transcription factors has been reported to increase yields under water-stress conditions, but such increases are generally not maintained under optimal conditions.21 Monsantos drought-tolerant (Genuity DroughtGard) corn hybrids are based on the introduction of bacterial chaperone genes.22 Fortunately, research into drought stress tolerance in wheat and other grains continues apace, although no drought-tolerant varieties have yet reached farmers.23
Real progress on crop yield is slow. What stands in the way is that we have so limited an understanding of how plants work at the molecular level. At every level of analysis, organisms are redundant networks of interconnected proteins that adjust their manifold physical and enzymatic interactions in response to internal signals and external stimuli, then send messages to the information storage facilities (DNA) to regulate their own production and destruction rates.
As well, many genes are present in families of between two and hundreds or thousands of similar members, making it difficult to determine either the function or the contribution of any given member to a complex trait such as stress tolerance or yield. That said, gene family functions are identifiable and some, such as transcription factor genes, encode proteins that influence multiple other genes, making them among the likeliest candidates for manipulation. Indeed, studies on the genetics of domestication often point to changes in transcription factor genes.24
But while there have been reports that constitutive overexpression of single transcription factor gene can increase grain yield in both wheat and maize, none appear to have been commercialized yet.25 The challenge of developing a yield-improved variety by simply overexpressing transcription factor genes is illustrated by a recent report from Corteva.26 It describes a tour-de-force involving generation and testing of countless transgenic plants to identify a single transcription factor gene, ZMM28, that reproducibly increased yield when incorporated into 48 different hybrids and tested over a 4-year period in 58 locations.26
Getting there by a different route
Might gene-editing facilitate the task of generating and identifying yield-enhancing genetic variation? While the CRISPR/Cas toolkit is growing at dizzying speed, its utility in crop improvement has so far been limited to the simple traits controlled by individual genes, albeit including multiple alleles.1,27
Crop domestication and plant breeding have vastly narrowed genetic diversity because the very process of selecting plants with enhanced traits imposes a bottleneck, assuring that only a fraction of the ancestral populations genetic diversity is represented in a new elite variety. This, in turn, limits what can be done by mutagenizing existing elite varieties, a process that is also burdened with the necessity to eliminate deleterious mutations through back-crossing.
But to widen the genetic base and to modify genes that contribute to quantitative traits, it is still first necessary to identify the genes that contribute to agronomically important traits. Identifying such genes is currently a slow and tedious process of conventional and molecular mapping.28 A recent report describes a method for combining pedigree analysis with targeted CRISPR/Cas-mediated knockouts that promises to markedly accelerate the identification of the individual contributing genes in the chromosomal regions that are associated with quantitative traits, technically known as quantitative trait loci (QTLs).29
Even as the QTL knowledge gap narrows, gRNA multiplexing is extending the power of SSNs to understanding and modifying complex traits in crop plants. For example, using multiplexed gRNAs, Cas nuclease was simultaneously targeted to three genes known to be negative regulators of grain weight in rice.30 The triple mutants were reported to exhibit increases in the neighborhood of 25% in each of the three grain weight traits: length, width and thousand grain weight.
In another study, 8 different genes affecting rice agronomic traits were targeted with a single multiplexed gRNA construct and all showed high mutation efficiencies in the first generation.31 Conversely, it has been reported that editing the same QTLs gives different outcomes in different elite varieties, improving yield in some but not other.32
Mutations affecting the expression of regulatory genes, such as transcription factors genes, account for a substantial fraction of the causative genetic changes during crop domestication.33 Multiplexed gRNAs constructs targeting cis-regulatory elements (CREs) have been used to generate large numbers of allelic variants of genes affecting fruit size in tomato, mimicking some of the mutations accumulated during domestication and breeding of contemporary tomato varieties.34
Knowledge of domestication genes can also be used to accelerate domestication of wild plants that retain traits of value, such as salt tolerance, as reported for tomato.35 This opens the possibility of rapidly domesticating wild species better adapted to the harsher climate conditions of the future.
While the above-described advances have been based on the CRISPR/Cas-mediated deletions, approaches to more precise sequence editing are developing as well. While Cas-generated cuts in the DNA are most commonly repaired by the non-homologous end joining pathway (NHEJ), the less frequent homology-directed repair pathway (HDR) has been shown to edit sequences at useful frequencies using Cas-gRNA ribonucleoprotein complexes.15,36
As well, mutant Cas9 proteins lacking nuclease activity have been fused with base-editing enzymes such as cytidine and adenosine deaminases to direct gene editing without DNA cleavage.37,38 This approach can change single base pairs precisely in both coding and non-coding regions, as well alter mRNA precursor processing sites.38 Finally, the sequence targeting properties of the CRISPR-Cas system can be used to deliver other types of hybrid proteins to target sequences to regulate gene expression and DNA methylation.27
In sum, the many variations on gene editing now developing hold the promise of revolutionizing crop breeding, prompting several colleagues to whimsically title a recent review of CRISPR/Cas-based methodology: Plant breeding at the speed of light.39 And indeed, the new methods make it possible to replace chemicals with biological mechanisms in protecting plants from pests and disease, as well as increase their resilience to stress.
That said, extraordinary progress in increasing grain yields has already been accomplished by what are now considered to be traditional breeding methods and increased fertilizer use. Further improvements continue, but will likely be harder won than the many-fold increases in corn, wheat and rice yields of the last century and its Green Revolution. But there is a persistent disconnect between what can be done to accelerate plant breeding using the new gene-editing toolkit and what is actually being done by both the public and private sectors to get varieties improved by these methods out to farmers.
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Nina V. Fedoroff is an Emeritus Evan Pugh Professor at Penn State University