A Guide to Microorganisms in Plant Biotech for Investors explains the science of CRISPR, the role of microorganisms in plant biotech and particularly how the evolution of these related systems are destined to ensure long term continued food security here in Australia and worldwide.
The aim of this case study is to summarise findings from a document review carried out based on several critical topics outlined by 'Case study topics - Microorganisms in Plant Biotechnology - BVB305 2018', to try and help potential investors who would like to educate themselves further in this field. A wide variety of sources including; scientific peer review literature, media, industry, books and commentary articles were considered. The purpose of this is to evaluate the pros and cons of using microorganisms or associated systems in plant genetic modification to address the food security challenge.
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Introduction
Farmers from around the world face real challenges to producing food because plants are under constant stress from factors like climate change, drought, disease, and pests. These are ever evolving growing conditions coupled with rapid population growth and changing diets, which represent concerns about long-term food security and the preservation of our global environment. Agriculture must respond with timely solutions to these urgent needs. It is estimated that by 2050, the human population will reach 10 billion, and global food production needs to increase by 60-100% (FAOSTAT 2016).
The primary finding in this case study is that CRISPR-Cas(Clustered Regularly Interspaced Short Palindromic Repeats) shows significant value-added potential for crop improvement applications. For agriculture, it offers a more targeted way to develop healthy plants and help the farmer produce more and better food with fewer resources, by selecting for characteristics such as higher yields, tolerance to drought, longer shelf life or better nutrition (Quinlan, 2018). Based on a natural defence, CRISPR can precisely improve a plant without incorporating DNA from another species, a real-world advantage when seeking regulatory approval compared with transgenic crops.
DNA is the instruction manual for the growth and development of all living organisms. In response to common internal and external stresses breaks and repairs of DNA strands routinely happen through natural cellular processes (Clancy, 2008). Over the past few decades, scientists have developed a deep understanding of the genetic and corresponding physical attributes within plants. With this knowledge, scientists can apply CRISPR-Cas to direct DNA breaks and repairs to create specific outcomes. Like a gene edit to remove DNA associated with pest susceptibility, conferring enhanced pest resistance in agriculturally important plants is one more way CRISPR-Cas can help address the world's food security challenge.
Types of microorganism based gene editing methods
We described in our primary findings, comparing the current genetic modification (GM) techniques, it seems CRISPR generated plant products show the best investment potential. Awareness in the advantages and disadvantages of using alternate GM methods will prove useful should investors look to predict the way CRISPR modified plant products could potentially capture a greater share of the agricultural marketplace. Mainly three classes of microorganism based gene editing methods are routinely employed those are; ZFN, TALEN and CRISPR.
ZFN – Zinc Finger Nucleases. They combine a non-bacterial artificial zinc finger array, made up of a chain of proteins with an enzyme FOK1 attached. A derivative of bacteria, the Fok1 enzyme is an endonuclease; therefore it cuts DNA with precision at certain sequences. With the ZFN in place on both strands of the DNA, the Fok1 molecules create a dimer, activating the enzyme and the mid-section is cleaved, creating a double-stranded break (DSB). The disadvantage to ZFNs is on the price per kit $3200 as per Sigma-Aldrich, associated with engineering the artificial zinc finger array (Aldrich, 2019).
TALENS - Transcription activator-like effector nucleases are fusion proteins of a bacterial TALE protein and FokI endonuclease. TAL effectors are proteins excreted by the bacteria Xanthomonas to regulate the transcription of rice genes when attached to nucleases; they are called TALENS (White, 2016). The way they cleave DNA is very similar to ZFNs. TAL effectors bind to DNA. However, they recognise individual nucleotides rather than triplets (Yeadon, J. 2019). The partner molecule binds to the other strand, and the attached Fok1 enzyme cuts the strands resulting in a DSB. The way TAL effectors bind DNA is that one subunit binds one nucleotide and subunits don't seem to interfere with each other as can be problematic with ZFNs (Yeadon, J. 2019). Engineered nucleases let researchers put changes in where they want them. There are still some improvements to be made; for example, the accuracy of the exact change varies while some nucleases can be hard to make. In comparison, TALENS are less complicated then ZFN for scientists to design and synthesise. TALENs reflect this fact as the particular cost seen can range from $350 - $1500 as per Thermofisher website, however it can vary a fair bit dependant on the type of kit and or specific application.
CRISPR – Clustered Regularly Interspaced Short Palindromic Repeats were first discovered by Japanese molecular biologist Ishino Yoshizumi in 1987 as part of an adaptive immune system in bacteria used to combat viruses (Ishino, Krupovic & Forterre, 2018). When viral DNA enters the bacteria, a Cas complex recognises the DNA and cleaves it into small fragments which are then inserted into the CRISPR locus as spacers between repeat sequences. Spacers in this sense, are stored as a molecular signature of viral DNA (Ishino, Krupovic & Forterre, 2018) (GALLEGOS, 2019).
Composed of gRNA (guide RNA) and Cas9 endonuclease, the gRNA has a complimentary sequence that matches with DNA at a precisely selected location (Ishino, Krupovic & Forterre, 2018). Part of which makes CRIPSR so useful is the fact this location can be controlled by altering the gRNA sequence (Jaganathan, Ramasamy, Sellamuthu, Jayabalan & Venkataraman, 2018). The Cas9 unwinds the DNA and helps pair the gRNA with the corresponding base pair sequence and then cuts the DNA breaking the DS (Carroll, 2017).
The observation that CRISPR-Cas9 system can make DSB on target DNA sequence guided by simple RNA DNA sequence complementarity inspired scientist to engineer this system to perform genome engineering in Eukaryotes (Carroll, 2017) (Komor, Badran & Liu, 2017). In 2013 2 scientific papers published simultaneously from George Church and Fungtanse Lab demonstrated the ability of CRISPR-Cas9 system to perform genome editing in Eukaryotic cells. According to, Borrelli, Brambilla, Rogowsky, Marocco & Lanubile, 2018 'Recently, all the other site-mediated methods such as ZFNs, TALENs have made way for CRISPR/Cas9 due to the ease design, ability to implement, a higher success rate, much better versatility with less expense.' A significant advantage to CRISPR-Cas is on the price per reagent typically a Cas9 endonuclease with gRNA costs as little as ~$200 as per Sigma-Aldrich (Aldrich, 2019).
Repair methods – Limitations and opportunities
The DSB created by each of these mechanisms is corrected using the cells own repairs methods. By default, if no homologous DNA is present, the cell uses the error-prone non-homologous end joining method (NHEJ) leading to knock-outs. Here ends of the DNA are literally ligated back together which in turn introduces small insertion or deletion (INDEL) mutations, which can alter gene function; researchers can use these INDELS to better understand gene control (Sonoda, Hochegger, Saberi, Taniguchi & Takeda, 2006). In contrast, providing a repair template is present; the cell can use homology-directed repair (HDR). The template sequence should closely match that of the area that surrounds the DSB and so is much more accurate, meaning that specific sequence changes can be introduced onto the desired gene (Sonoda et al., 2006). With CRISPR it is possible to engage a modified Cas9 like SpCas9n to allow knock-in via HDR, or even a CRISPR-Cpf1 system in rice crops derived from Lachnospiraceae bacterium developed to increase target specificity and reduce off-target cleavage (Yin et al., 2017). It is precisely cutting edge development like these types where potential investors should focus on as CRISPR science evolves with plant biotech.
The role of microorganisms in plant biotech from the laboratory to the field
There are different methods scientists can implement microorganisms and related systems to transport the DNA of interest in a newly transformed plant cell. To direct the activity of the new gene desired in plant cells scientists select a promoter start material commonly derived from a viral microorganism called the cauliflower mosaic virus (camv35s). While others have described viruses for this purpose like Figwort mosaic virus (FUAS35SCP), Mirabilis mosaic virus (MUAS35SCP) and Cotton leaf curl Burewala virus (CLCuBuV) (Patro, Kumar, Ranjan, Maiti & Dey, 2012) these may show plant species-specific characteristics that with the right development look like areas with investment potential. At this time point, Cmv35s remains the general promoter of choice with scientists since it has the advantage of abundant production of transgenic protein in a wide variety of applications (De Ponti, 2013) (Khan, Abdin & Khan, 2015).
With the large number of DNA required to make a plant, the easiest way is to use bacterial cell machinery to produce accurate copies. Plasmid DNA can be replicated using a bacteria called Agrobacterium tumefaciens. It is the only known prokaryotic organism with the natural ability to transfer DNA to eukaryotic cells (Ream 1989; Tzfira and Citovsky 2008). The Agrobacterium transfers a segment of DNA from a plasmid into a plant cell, causing the plant cells to grow, thus causing the tumor and produce a food product for the Agrobacterium called opines. Tumor inducing (Ti) plasmid, a considerable size it contains 200 genes and over 200 000bp (Hwang, Yu & Lai, 2017). Like all plasmids, it contains an origin of replication so that the plasmid can be copied. There are a set of virulence genes or bacterial type IV secretion system (T4SS) required from infecting and transferring DNA to the plant. This is separate operon containing genes required to metabolise opines the plant will make for food for the bacteria. A section of the plasmid contains a sequence of DNA that will be transferred to the plant. This segment is called transfer DNA (T-DNA); T-DNA has two main parts. One bit contains genes to trigger tumour formation, and another bit contains the genes to make the opines to feed the Agrobacterium (Hwang, Yu & Lai, 2017).
To use this plasmid to make transgenic plants, the genes that are harmful to the plant are removed and the genes that are to be transferred into the plant are added. Once the opine synthesis genes as well as the tumor-inducing genes are removed, the gene for the trait as well as the promoter are put in this place (Brewer, Hird, Bailey, Seal & Foster, 2018). Commonly a very strong promoter such as camv35s is preferred to ensure that the production of the desired gene is high (Khan et al., 2015). A selectable marker is introduced with the bacterial promoter to allow for the selection of Agrobacterium that contains the Ti plasmid. The virulence genes (T4SS) remain, as they are necessary to transfer the T-DNA from the Agrobacterium to the plant.
The Ti plasmid transfer from the Agrobacterium involves the expression of a number of genes in the Agrobacterium cell. Because living plant cells release a variety of small molecules, Agrobacterium detects these then triggers the expression of a transcription factor that initiates the transcription of the virulence genes on the Ti plasmid (Hwang, Yu & Lai, 2017). One of the gene products leads to the production of the T-DNA sequence, which then attaches another virulence protein which shuttles the plasmid itself into the plant cell. The T-DNA incorporates randomly into the chromosomal DNA. A major advantage of the bacterial entry method is a high level is target specificity, in comparison with the gene gun method, for example, meaning much less damage to the cell and no chance of incorporation into the chloroplast or mitochondrial DNA (Gao & Nielsen, 2013).
To select cells containing the plasmid, the addition of an antibiotic resistance gene ensures that when bacteria are grown on plates containing the antibiotic, only the cells containing the plasmid will grow and ultimately produce GM plants. According to, Gay & Gillespie, 2005 'The nptll gene is one of the most frequently used selectable markers in plant cell modification alongside hph genes (Breyer, Kopertekh & Reheul, 2014) The nptII is derived from Escherichia coli (E. coli) strain K12 (Beck et al., 1982) encodes a neomycin enzyme that inactivates the aminoglycoside antibiotics neomycin, kanamycin, and paromomycin.' Alternatively, reporter genes code for molecules that can be readily identified visually or by biochemical assay (Breyer, Kopertekh & Reheul, 2014) performs a similar function. The uidA gene from E.coli encodes the enzyme β-glucuronidase, which enables E.coli to metabolise β-glucuronides as a source of carbon and energy (Gilissen et al., 1998). In this process which kills plants cells, using a substrate of the enzyme which produces a coloured product when cleaved (Jefferson and Wilson, 1991).
Lately, scientists have developed CRISPR into a fast new method to label without denaturing the cell, called RNA guided endonuclease – in situ labeling (RGEN-ISL). The tool was developed to visualise plant genomes based on a two-part guide RNA with a recombinant Cas9 endonuclease complex. It delivers two distinct advantages compared to GUS reporter genes such that it does not require a special transformation method nor does it require DNA denaturation (Ishii et al., 2019).
Now a transformed plant cell, under sterile conditions the plant cells are grown on media that contains both the antibiotic for selection and plant hormones. The antibiotic ensures plant cells selectively transformed will grow and the plant hormones allow for the generation of an entire plant from just a few plant cells. After the plant has developed both the root and shoot portions of the plant, it can be transferred to regular soil and the new properties of the plant can be tested.
Conclusion and recommendations
This Guide to Microorganisms in Plant Biotech for Investors has explained the science of CRISPR, the role of microorganisms in plant biotech and particularly how the evolution of these related systems are destined to ensure long term continued food security here in Australia and worldwide. CRISPR is the latest genome editing method scientists have developed with the potential to address real-world food security concerns. The primary finding in this case study is that CRISPR-Cas shows significant value-added potential for crop improvement application. Compared with ZFN and TALEN systems, CRISPR offers several advantages like target design simplicity, efficiency, and low cost.
We have combined a document review from a wide variety of sources to help inform the potential investor and evaluate the pros and cons of using microorganisms or associated systems in plant genetic modification to address the food security challenge. We identified several areas with significant potential for investment around improved specificity and reduced off-site effects as well as better promoters tailored to specific plant species for higher efficiency and even new molecular imaging applications. A recent announcement early 2019 by the Australian Office of the Gene Technology Regulator (OTGR) has indicated a move towards the deregulation of gene editing like CRISPR. This should provide investors some clarity around the Australian biotech landscape for investment certainty within the field.
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