Genome Engineering

Workpackage D: Genome Engineering

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 

RNA­-guided Cas9-­mediated targeted mutagenesis

OpenPlant has demonstrated RNA­-guided Cas9-­mediated targeted mutagenesis or gene deletion in multiple species including:

• Nicotiana benthamiana

• Arabidopsis

• 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).

• Marchantia polymorpha (Haseloff Lab and Schornack Lab)

Plants  with disrupted selection cassettes have been created to enable efficient recovery of targeted insertion events. Simultaneous delivery of nucleases and the repair template to protoplasts resulted in the recovery of callus with targeted insertions on selective media.

standardized toolkit for genome engineering

Oleg Raitskin (Patron lab) has developed a suite of Cas nuclease variants and an associated toolkit for targeted mutagenesis and gene deletion in multiple plant species (Raitskin et al, 2019). These Cas variants have been compared for specificity and efficiency using next­-generation sequencing technologies and digital­ droplet based PCR assays . Methods for protoplast delivery developed in this study were also applied to potato (Workpackage G) in collaboration with Aytug Tuncel in the Smith lab (JIC) (Tuncel et al., 2019).

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.

Training

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, 2016; Raitskin and Patron, 2016)

Chloroplast manipulation

Yeast artificial chromosome vectors as plant shuttle systems

Hierarchical DNA assembly methods are a necessary part of genome construction and modification. Large-scale assemblies require specialized cloning vehicles like artificial chromosomes for construction and transfer of genetic circuits to target organisms. Recent developments are producing efficient tools for modifying chromosomal sequences in situ. Work is ongoing 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.  

Fluorescent reporters 

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. Our latest results have been included in Sauret-Gueto et al. (2020).

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).

Publications

Hayta S, Smedley MA, Clarke M, Forner M, Harwood WA (2021) An Efficient Agrobacterium-Mediated Transformation Protocol for Hexaploid and Tetraploid Wheat. Curr Protoc. 1(3):e58. doi: 10.1002/cpz1.58.

Smedley MA, Hayta S, Clarke M, Harwood WA (2021) CRISPR-Cas9 Based Genome Editing in Wheat. Curr Protoc. 1(3):e65. doi: 10.1002/cpz1.65.

Sauret-Güeto S, Frangedakis E, Silvestri L, Rebmann M, Tomaselli M, Markel K, Delmans M, West A, Patron NJ, Haseloff J. (2020). Systematic tools for reprogramming plant gene expression in a simple model, Marchantia polymorpha. ACS Synth. Biol. 9, 4, 864–882 https://doi.org/10.1021/acssynbio.9b00511

Debernardi JM, Tricoli DM, Ercoli MF, Hayta S, Ronald P, Palatnik JF, Dubcovsky J (2020). A GRF-GIF chimeric protein improves the regeneration efficiency of transgenic plants. Nature Biotechnology 38: 1274–1279 https://doi.org/10.1038/s41587-020-0703-0

Patron, N. J. (2020) Beyond natural: synthetic expansions of botanical form and function. New Phytologist 2: 295-310 https://doi.org/10.1111/nph.16562

Nimmo IC, Barbrook AC, Lassadi I, Chen JE, Geisler K, Smith AG, Aranda M, Purton S, Waller RF, Nisbet RER, Howe CJ (2019) Genetic transformation of the dinoflagellate chloroplast. eLife. 2019; 8: e45292. doi: 10.7554/eLife.45292

Raitskin O, Schudoma C, West A, Patron NJ. (2019) Comparison of efficiency and specificity of CRISPR-associated (Cas) nucleases in plants: An expanded toolkit for precision genome engineering. PLoS One. 14(2):e0211598. doi: 10.1371/journal.pone.0211598.

Tuncel A, Corbin KR, Ahn‐Jarvis J, Harris S, Hawkins E, Smedley MA, Harwood W, Warren FJ, Patron NJ, Smith AM (2019) Cas9-mediated mutagenesis of potato starch branching enzymes generates a range of tuber starch phenotypes. Plant Biotechnol J. 17(12):2259-2271. doi: 10.1111/pbi.13137.

Crozet P, Navarro FJ, Willmund F, Mehrshahi P, Bakowski K, Lauersen KJ, Pérez-Pérez ME, Auroy P, Gorchs Rovira A, Sauret-Gueto S, Niemeyer J, Spaniol B, Theis J, Trösch R, Westrich LD, Vavitsas K, Baier T, Hübner W, de Carpentier F, Cassarini M, Danon A, Henri J, Marchand CH, de Mia M, Sarkissian K, Baulcombe DC, Peltier G, Crespo JL, Kruse O, Jensen PE, Schroda M, Smith AG, Lemaire SD (2018) Birth of a Photosynthetic Chassis: A MoClo Toolkit Enabling Synthetic Biology in the Microalga Chlamydomonas reinhardtii. ACS Synth Biol. 7(9):2074-2086. doi: 10.1021/acssynbio.8b00251.

Salzman AE (2017) Controlling Relative Gene Expression Using Orthologous Regulatory Elements: Exploring Future Prospects of Tuneable Control for the Manufacture of Natural Products. Masters thesis. University of East Anglia.

Volpi e Silva N and Patron N (2017) CRISPR-based Tools for Plant Genome Engineering. Emerging Topics in Life Sciences doi: 10.1042/ETLS20170011

Raitskin O, Patron NJ, (2016). Multigene Engineering with RNA guided Cas9 Nuclease. Current Opinion in Biotechnology 37; 69-75 https://doi.org/10.1016/j.copbio.2015.11.008

Belhaj K, Chaparro-Garcia A, Kamoun S, Patron NJ, Nekrasov V (2015). Editing plant genomes with CRISPR/Cas9. Curr Opin Biotechnol. 32:76-84. doi: 10.1016/j.copbio.2014.11.007.

Lawrenson T, Shorinola O, Stacey N, Liu C, Østergaard L, Patron NJ, Uauy C, Harwood, W (2015). Induction of targeted, heritable mutations in barley and Brassica oleracea using RNA guided Cas9 nuclease. Genome Biol. 16:258. doi: 10.1186/s13059-015-0826-7.