• Research

Genome engineering

Using programmable nucleases to make targeted modifications to plant genomes.

Project summary.

Led by: Patron Group

Start date: 31 July 2014

End date: 2 September 2019

Duration: 61 months

Funder: BBSRC

Value: 

£11,787,968  (total to the whole consortium, led by Sir David Baulcombe, UCam)

£594,132 (total to the whole grant, led by Prof. Wendy Harwood, JIC)

Grants:

BBSRC Synthetic Biology Research Centre ‘OpenPlant’ Award (BB/L014130/1)

BBR Targeted gene knockouts in crops using RNA-guided Cas9 nuclease BB/N019466/1

We are using genome engineering technologies to make plants into efficient factories for the biosynthesis of vaccines and therapeutics and also to improve crops for increased production and nutritive value.

Humans have been changing the genomes of plants for thousands of years. The domestication of crops by selective breeding fixed desirable traits, for example for high yield, but in doing so reduced genetic diversity. Breeding and mutagenesis using radiation and chemicals are still widely used to increase the variation in plant genomes, allowing the selection of new and desirable traits. In the last few years, new tools have been invented that allow us to target specific genes for which we either already know the function of, or would like to learn more about. We can now create mutations in these genes, without affecting the rest of the genes in the plant. 

We have shown that we are are able to use genome engineering tools to introduce precise changes in a target gene to obtain plants with a desired characteristic and recover plants that contain no foreign DNA. We are currently working on improving the efficiency and specificity of the tools. We are also aiming to engineer plants species used for the biosynthesis of proteins and small molecules such as vaccines and therapeutics to improve the yield and purity and to work with collaborators to study specific genes in crop plants.

Details.

We are using RNA-guided Cas proteins from the CRISPR (Clustered Regulatory Interspaced Short Palindromic Repeats) loci found in bacterial adaptive immune systems to induce breaks in specific sequences of DNA. The use of RNA-guided Cas9 for the engineering of both single and multiple genes has been demonstrated in a number of plant species in the past few years, Although Cas9 is a large monomeric protein, the single guide RNA (sgRNA) that directs it to a specific DNA target sequence is small and easy to reprogram. Using a modular cloning system we are able to easily produce constructs with several sgRNAs to target multiple endogenous gene sequences. 

We have shown that we are are able to make transgene-free plants with just a few base-pair changes in a target gene and also to make larger deletions. We are currently working on improving the efficiency and specificity of our toolkit, novel methods for quantification of mutagenesis, and applying our tools to specific genetic loci in a number of different plant species.

Tools.

The following molecular tools are available from AddGene. These were used to construct circuits for Cas9-induced targeted mutagenesis in barley (Hordeum vulgare) and/or Brassica oleracea using the the Golden Gate MoClo Plant Toolkit and additional elements from the Golden Gate MoClo Plant Parts Kit.

#68262 pICSL90003  Level 0: PROM U6 (Triticum aestivum)

#68261 pICSL90002  Level 0: PROM U6-26 (Arabidopsis thaliana)

#68257 pICSL12009  Level 0: PROM Ubi (Zea mays)

#50270 pICSL12006  Level 0: PROM CsVMV (Cassava Vein Mosaic Virus)

#68260 pICSL80037  Level 0: CDS neomycin phosphotransferase II (Escherichia coli)

#68259 pICSL80036  Level 0: CDS hygromycin phosphotransferase II (Escherichia coli)

#50339 (from the Kamoun lab) Level 0: CDS Cas9 (Streptococcus pyogenes)

#46966 (from the Kamoun lab) Level 0: sgRNA scaffold

#68263 pICSL11059  Level 1, Position 1: CaMV35s:TMV-Omega_hptII with intron_CaMV35s

#68252 pICSL11055  Level 1: CaMV 35S_nptII_nos

#68264 pICSL11060  Level 1, Position 2: CsVMV_Cas9_CaMV35s

#68258 pICSL11056  Level 1, Position 2: ZmUbi_Cas9_CaMV35s

Collaborators.

The OpenPlant Consortium

OpenPlant is a collaborative initiative between the University of Cambridge, the John Innes Centre and the Sainsbury Laboratory in Norwich. The initiative promotes interdisciplinary exchange, open technologies for innovation and responsible innovation for sustainable agriculture and conservation. Dr Nicola Patron is leading a Work Package in genome engineering to create a suite of efficient and precise tools for different plant species and collaborating with other groups within the consortium to apply this technology to different plant species.

Professor Wendy Harwood

Prof. Harwood leads the Crop Transformation Group, BRACT at the John Innes Centre, that develops and delivers efficient crop genetic modification and genome editing technologies.  Wendy is leading the application of genome editing technologies to crop species.

Impact statement.

Genome engineering technologies are transforming fundamental research in every field. Compared to genetic modification using transgenes, genome editing can result in just a small mutation in a target gene and no other changes in the plant genome. The availability of efficient and specific genome engineering toolkits for different plant species will allow the yield and purity of vaccines and therapeutics produced in plants to be improved and will also have wider benefits to agricultural productivity as new traits are demonstrated in crops and feed through to breeding programmes.