By Steven Burgess and Iulia Gherman
Synthetic biology certainly includes plants, but the field of plant synthetic biology is less developed compared to model heterotrophs or mammalian applications. But plants are important, too important in fact. A recent PNAS paper estimates the amount of global biomass and the contributions of different taxa, and plants are by far the largest contributors to global biomass and dominate terrestrial ecosystems. The last few years’ developments have promised to make plant synthetic biology more approachable, and the interest of plant researchers in synthetic biology is growing, as reflected by the plant synthetic biology conferences and special issues in plant journals. In this post our former editor Steven Burgess and Iulia Gherman, both talented plant researchers, share their thoughts on the present and future of plant synthetic biology.
Read more: Why Plants? – Feynman and Flowers
While synthetic biology in bacteria, yeast and mammalian cells has advanced by leaps and bounds, plant synthetic biology was off to a slower start. Designing circuits in plants is complicated by the polyploid nature of some genomes, the difficulty of transient transformation, the inability to control the placement of transgenes, and the long generation times, to name a few major challenges. On the regulatory side of things, special licences are necessary to produce and grow genetically modified plants, while field trials for crops require additional permissions by national authorities.
However, progress has been made in a number of key areas, and the tools and technology are catching up with their bacterial and mammalian counterparts. Recent advances include the ability to characterise and screen circuit parts in protoplasts in a high-throughput manner, targeted genome insertions via CRISPR-Cas9, and improvements in plant growth through speed breeding. Over the past decade, the development of modular cloning techniques has transformed the rate of progress in plant science. A host of systems have been used, including Gibson assembly, golden-gate cloning, and recently Loop assembly, which incorporates unique nucleotide sequence (UNS) cloning for mixing of parts from different systems.
These developments have been aided by efforts to implement community standards in plant synbio, an open material transfer agreement to facilitate sharing of resources, and a growing collection of modular parts being created by ENSA, the OpenPlant, C4 rice and RIPE projects, some of which are available in repositories like Addgene (or for Marchantia on MarpoDB).
These advances have greatly facilitated crop improvement efforts. For example, the C4 rice project, which aims to introduce multiple genes to manipulate plant morphology and biochemistry, started in the mid-2000s. At the time you could make one construct per gene, and in order to stack these into one variety an individual transgenic line had to be created for each, followed by an elaborate crossing process which took years. Then with the development of golden-gate cloning, a single multi-gene construct was created and inserted into rice, slashing the time to analysis as it required only one round of transformation.
The new technological developments pave the way for introducing genetic circuits to perform various functions in plants. One engineering dream is to synthesise a completely in vivo controller (with both sensor and actuator functions performed by the cells) to maintain constant levels of some quantity, be it genes, burden, ribosomes, in the face of noise and perturbation. This has been achieved in theory numerous times – and recently also in practise in E. coli. It is only a matter of time before synthetic controller designs are implemented in vivo in plants.
However, when it comes to plants, generating constructs is only part of the challenge: the process of delivering DNA into species of interest is a major bottleneck in addition to issues associated with the precise manipulation of plant genomes. Beyond model species like Arabidopsis thaliana, which can be easily transformed by floral dip, transgenic plants are commonly generated through a process of tissue culture. This is can be a long and expensive process; it is genotype dependent and success rates vary widely.
Currently, after generating transgenic lines it is necessary to go through 2 or 3 life cycles to end up with homozygous transformants. Position effects from random integration of transgenes, silencing and mosaicism are all issues. A rough figure of 30 plants should ideally be screened to get 3 single insertion lines for analysis. All this requires generating and maintaining vast numbers of calli (regenerated plant tissue) on expensive hormone mixtures. For those species where a commercial facility offers a transformation service, it will typically cost in the region of several thousand pounds for a single transgenic line.
What’s more, unfortunately even if there is a transformation procedure for a crop, elite cultivars are often recalcitrant to transformation, meaning genes have to be added to a donor line which must be backcrossed to produce a commercial variety.
Make the art of plant transformation a routine process
So what can be done to address some of these issues? Is it possible to apply principles of automation and standardization to transformation and breeding? Several potential solutions lie in the past.
In order to overcome the issues associated with random integration of DNA constructs, research has produced a series of recombinase systems for site specific integration of transgenes, thereby providing consistent expression patterns. Wider use could improve reproducibility. Alternatively, the Voigt lab recently demonstrated a CRISPR based system for standardizing expression levels of transgenes in E. coli, the principle of which may be useful for adaptation to plant research.
There is something of an art to tissue culture transformation, and regeneration of calli, it is manual and labour intensive. However, there is also a long history of regenerating plants from protoplasts – single cells released by enzymatic digestion of tissue, with reports of successfully regenerating whole plants from crops including maize and rice. The potential advantage of such an approach, is that it affords the possibility of using automated workflows commonly used in engineering microorganisms. The first steps of such a hypothetical pipeline have recently been demonstrated, including using a robotic platform for protoplast transformation, or, in a project I was involved with, using microfluidics to encapsulate and sort protoplasts based on the expression of a fluorescent transgene.
There is a long way to go but there are promising signs. Plant synbio will continue to benefit from developments in other fields, adapting techniques to address its unique challenges as well as careful stepwise improvement of existing systems (such as tweaking the origin of replication used in binary vectors to increase the frequency of single insertions). A phrase from physicist and CEO of CellFreeTech Tom Meany sticks with me – “if synbio is to be successful it needs to get really boring”, while I think there is always room for exciting innovations, I also believe there is a great deal of truth in the statement – a good dose of dull grunt work will go a long way.
New technologies for novel designs
Precise genome editing
A myriad of CRISPR-CasX tools are now available for synthetic biologists to take advantage of. One such variant on the original CRISPR-Cas9 includes the use of a “deactivated” or catalytically “dead” – dCas9 enzyme, whose nuclease activity is disabled. This is usually fused to DNA-binding modules, making it capable of transcriptional activation or repression. The gDNA then directs these Cas9 transcriptional regulators to specific promoters, such as this implementation in the genome of N. benthamiana. A newly identified nuclease, Cas13, binds to and cleaves single-stranded RNA molecules (as opposed to double stranded DNA). This property is being exploited to combat plant RNA viruses, implement post-transcriptional regulation superior to RNAi, image translocation of mRNA molecules with dCas13, and for base editing of mRNAs.
Gene drives, the real game changer
Another exciting application of CRISPR-Cas9 is the gene drive, which has been lauded as a game-changer for pest control. A gene drive breaks inheritance rules by getting passed on to the majority of progeny, rather than 50%, given one parent with the gene drive in their genome and one wild-type parent. The beauty of a gene drive lies in its simplicity. All it requires is a plasmid with a cassette containing the Cas9 gene, a gRNA that targets a particular gene in the host and homologous regions flanking the cassette, enabling its insertion into the cleavage site of the gene. Once this cassette is inserted into the genome, it can continue propagating itself in this manner into wild-type copies of the gene.
Would it be possible to harness the main benefit of gene drives – >90% transmission rate to the next generation – in plants, especially polyploid crops, in order to make homozygous knockouts in a single generation?
Regulation of edited plants
The potential and growth of new breeding technologies (including CRISPR-CasX) is staggering. However, progress very much depends on the regulatory environment. Following a lengthy debate regarding the classification of CRISPR technologies as GM or mutagenesis techniques, the European Court recently ruled that CRISPR-modified crops are to be classed as GM. This is considered a major drawback for European plant synthetic biology as funding will likely dry up for a technology with endless applications if they cannot actually be put into practice outside a laboratory. There have been calls to make the regulation of GMOs product-based rather than technology based instead, similar to the regulatory system in Canada.
In the summer of 2017, CRISPR-y cabbage, made (and grown!) by Stefan Jansson was served in Gothenburg as part of the SEB meeting “New breeding technologies in the plant sciences – Applications and implications in genome editing” conference meal. It was delicious. Today this would not be possible.
Steven Burgess is a visiting scholar at the University of Illinois, working on the RIPE project
Iulia Gherman is a final year PhD student working on re-wiring, in silico and in vivo, gene regulatory networks in Arabidopsis.
Declaration: Steven Burgess was previously employed by Prof Julian Hibberd who has funding from the C4 rice project. He received grant money from the OpenPlant Project to pursue a research project and is currently employed on the RIPE project as a visiting schola