• Research

Plant Genome Engineering

We are designing and applying molecular tools that can make precise deletions, insertions and edits in plant genomes.

Project summary.

Led by: Patron Group

Start date: July 2014

End date: September 2019

Duration: 61 months

Funder: BBSRC

Value: £12,391,458

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

Humans have been changing the genomes of plants for thousands of years. The domestication of crop plants fixed genes for desirable traits, for example for high yield, but in doing so reduced genetic diversity. Current crop improvement practices use several methods to increase genetic variation in plant genomes to develop new desirable traits. The most recent and precise of these are molecular tools that enable insertions, deletions or edits to be made in specific DNA sequences. This is known as 'genome engineering' tools. In this project, we have shown that we are able to use these tools in plants, including crop species to introduce precise changes in plant genes that result in expected changed. We are now working on improving the efficiency and specificity of the tools and applying them to useful traits.

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.

We are able to rapidly design and test several sgRNAs to induce precise changes in multiple genes at the same time. We have shown that we are able to make transgene-free plants with specific changes in a target gene. We are currently working on improving the efficiency and specificity of our toolkit and applying our tools to engineer useful traits and investigate the function of genes.

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

Publications.

Zinc-Finger Nuclease (ZFN)-mediated precision genome editing of an endogenous gene in hexaploid bread wheat (Triticum aestivum) using a DNA repair template

Ran Y, Patron N, Kay P, Wong D, Buchanan M, Cao Y, Sawbridge T, Davies J, Mason J, Webb S, Spangenberg G, Ainley W, Walsh T, Hayden, M, Plant Biotechnology (2018)

CRISPR-based tools for plant genome engineering

Volpi e Silva N and Patron NJ, Emerging Topics in Life Sciences (2017)

Multigene engineering in plants with RNA-guided Cas9 nuclease

Raitskin O, Patron NJ, Current Opinion in Biotechnology (2016)

Induction of targeted, heritable mutations in barley and Brassica oleracea using RNA-guided Cas9 nuclease

Lawrenson T, Shorinola O, Stacey N, Liu C, Østergaard L, Patron NJ, Uauy C, Harwood, W, Genome Biology (2015)

Editing plant genomes with CRISPR/Cas9

Belhaj K, Chaparro-Garcia A, Kamoun S, Patron NJ, Nekrasov V, Current Opinion in Biotechnology (2015)

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. In plants, they can be used to rapidly produce transgene-free plants with desirable genotypes, for example to remove genes for toxins and antinutrients, to improve nutritive value or to remove genes for disease susceptibility.

Related reading.