OpenPlant is developing editing tools and large scale gene assembly technologies to enable genome engineering across multiple species and in both nuclear and plastid genomes.
Molecular tools for targeted genome editing
The Patron lab has demonstrated RNA-guided Cas9-mediated targeted mutagenesis or gene deletion in multiple species including:
- Nicotiana benthamiana
- tomato (collaboration with Banfield Lab at John Innes Centre)
- potato (Aytug Tuncel; Smith Lab at John Innes Centre)
- barley (collaboration with Harwood, Uauy & Wulff Labs at John Innes Centre; Lawrenson et al, 2015)
- Brassica Oleracea (collaboration with Harwood lab at John Innes Centre; Lawrenson et al, 2015).
Plants with disrupted selection cassettes have been created to enable efficient recovery of targeted insertion events. Preliminary attempts to repair these cassettes by simultaneous delivery of nucleases and repair template have begun in Nicotiana tabacum, Nicotiana benthamiana, Arabidopsis and barley.
RNA-guided Cas9-mediated targeted mutagenesis in Marchantia has been demonstrated by Bernardo Pollack in the Haseloff lab and this has now also been established in the Schornack Lab.
Oleg Raitskin (Patron lab) has developed a suite of Cas9 variants and an associated toolkit for targeted mutagenesis and gene deletion in multiple plant species. These are currently being compared for specificity and efficiency using next generation sequencing technologies and digital droplet based PCR assays.
Assembly of plasmid vectors for targeted mutagenesis is being automated at the DNA foundry at the Earlham Institute (Patron lab). A protoplast assay for rapid assessment of constructs has been established for tobacco, Arabidopsis and barley.
An OpenPlant CRISPR/Cas9 workshop was held at the John Innes Centre in September 2015, cofunded by the Genomic Arabidopsis Resource Network (GARNet) and sponsored by Methods in Plant Biology. A special collection of methods on Plant Genome Engineering was published in conjunction with the meeting report (Parry et al., 2016).
Reviews and text book chapters on the use of CRISPR/Cas9 for plant engineering are also being produced from OpenPlant labs (e.g. Patron, 2016b; Raitskin and Patron, 2016)
Work is ongoing by Orr Yarkoni in the Ajioka Lab to construct yeast based systems for genome scale DNA assembly, editing and shuttling to plant systems. The 121kb Marchantia chloroplast genome is the first target for large DNA manipulation - its size is beyond the range of conventional plasmid cloning strategies, but is relatively small, easier to handle in vitro and and of great interest for metabolic engineering.
In the first stage of the work, the plastid genome annotation has been manually curated and updated. Restriction Enzyme engineering sites have been identified for designing a synthetic genome. Design of the DNA assembly strategy for the synthetic plastid genome is ongoing, as manual curation of the genome has identified areas that are complex to engineer.
Christian Boehm (Haseloff lab; Boehm et al., 2015) established plastid transformation in Marchantia and created the first fluorescent markers for liverwort plastid transformation. Fluorescent reporters with spectra ranging from 355nm excitation to 670nm emission, essentially from UV to near infrared, have now been selected and optimized using a codon optimization tool developed in the Ajioka Lab to adapt them for the specific requirements of the Marchantia plastid genome. These reporters are in the final stages of assembly and will be trialed in Marchantia in the coming months.
iGEM 2016 - InstaChlam
The 2016 Cambridge-JIM iGEM team, supported by OpenPlant, aimed to create a toolkit for algal chloroplast engineering, a process that holds great potential for producing everything from biofuels to edible vaccines both efficiently and in large quantities. In just ten weeks the team managed to generate a library of tested parts optimised for Chlamydomonas algae, build a gene gun for less than 1/100th of the current commercial price and design a genetic tool which helps achieve transformation of all DNA contained within a chloroplast (homoplasmy) in a much shorter time frame than previously possible. This work won them a Gold Medal and the 2016 Best Plant Synthetic Biology Prize (overgrad category).
Boehm, C.R., Ueda, M., Nishimura, Y., Shikanai, T. and Haseloff, J., 2015. A cyan fluorescent reporter expressed from the chloroplast genome of Marchantia polymorpha. Plant and Cell Physiology, p.pcv160. DOI: 10.1093/pcp/pcv160
Lawrenson, T., Shorinola, O., Stacey, N., Li, C., Østergaard, L., Patron, N., Uauy, C. and Harwood, W., 2015. Induction of targeted, heritable mutations in barley and Brassica oleracea using RNA-guided Cas9 nuclease. Genome biology, 16(1), p.258. DOI: 10.1186/s13059-015-0826-7
Parry, G., Patron, N., Bastow, R. and Matthewman, C., 2016. Meeting report: GARNet/OpenPlant CRISPR-Cas workshop. Plant methods, 12(1), p.6. DOI: 10.1186/s13007-016-0104-z
Patron, N.J., 2016a. Blueprints for green biotech: development and application of standards for plant synthetic biology. Biochemical Society Transactions, 44(3), pp.702-708. DOI: 10.1042/BST20160044
Patron, N.J., 2016b. Synthetic Plants. In Synthetic Biology Handbook (pp. 183-206). CRC Press.
Raitskin, O. and Patron, N.J., 2016. Multi-gene engineering in plants with RNA-guided Cas9 nuclease. Current opinion in biotechnology, 37, pp.69-75. DOI:10.1016/j.copbio.2015.11.008