DEPARTMENT OF ECONOMIC DEVELOPMENT, JOBS, TRANSPORT & RESOURCES Precise Trait Engineering in Wheat Using EXZACT TM Technology Dr Matt Hayden 24 September 2015
Jan 17, 2016
DEPARTMENT OF ECONOMIC DEVELOPMENT, JOBS, TRANSPORT & RESOURCES
Precise Trait Engineering in Wheat Using EXZACTTM Technology
Dr Matt Hayden24 September 2015
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How Wheat Is Genetically Modified
Mutagenesis
Exposing seeds to chemicals or radiation
Traditional Breeding
Crossing plants and selecting offspring
Number of Genes Affected
Many Difficult to assess Few
Desired gene(s) introduced with other
genetic material
Random changes in genome, usually
unpredictable
Desired gene(s) inserted into genome
Transgenics
Inserting or modifying genes using
recombinant DNA methods
*adapted from www.geneticliteracyproject.org
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Random insertion of foreign DNA into the genome.
Applications
– Add new gene; transgenics or cisgenics
– Switch off / down regulate native (endogenous) gene expression; RNA silencing
Limitation
– Level of transgene expression dependent on site of integration; need to generate multiple plants to find the one with optimal transgene expression for variety development, or to determine “average” transgene effect for research purposes
Conventional Genetic Engineering
Random location
Random insertion
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Precision Genome Editing
Targeted modification of a specific site in the genome.
Engineered nucleases are used to create a double stranded DNA break at a pre-determined position.
– Homing endonucleases / mega-nucleases
– ZFNs (zinc-finger nucleases)
– TALENs (transcription activator-like effector nucleases)
– CRISPR-Cas9 (clustered regularly interspaced short palindromic repeat-associated nuclease)
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Precision Genome Editing
Engineered nuclease induction of a double stranded break stimulates native cellular DNA repair that can be used to precisely modify the target locus.
Mutagenesis DeletionChromosomal
deletion
Gene editing Gene conversion
Transgene insertion
Target sequence
Foreign DNA
*adapted from Tzfira et al. (2012) Plant Biotech. J.
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Enabling EXZACT Technology in Wheat
Development of platform technology for implementation of EXZACTTM (ZFN-mediated) precision genome editing in bread wheat.
Triticum aestivum(AABBDD, 2n=42)
17 Gb genome
Approach1. Develop transient assay systems to investigate strategies and
explore construct designs for achieving precise integrative and non-integrative editing outcomes via EXZACT Delete, Edit and Add modes.
2. Establish a transformation system for generating wheat plants demonstrating EXZACT precision trait engineering.
Questions• Can sub-genome-specific ZFNs be designed for a polyploid species
that has 3 closely related but not identical sub-genomes? • Is there a design capability limit (i.e. minimum sequence window
size) for targeted ZFN design? • Is it possible to recover precision edited wheat plants with a
modified phenotype, or to modify a non-selectable trait locus?
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AHAS (EC 2.2.1.6) catalyses key steps in the synthesis of branched-chain amino acids: leucine, valine and isoleucine.
Group B herbicides inhibit AHAS activity, leading to plant death by amino acid starvation.
5 mutations in AHAS confer tolerance to AHAS-inhibiting herbicides.
A122T (6D) and S653N (6A,6B,6D) mutations have been characterised in wheat.
Acetohydroxyacid Synthase (AHAS)
AHAS selected as model gene for EXZACT trait engineering, with S653N as target mutation
*position in reference to Arabidopsis thaliana
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Approach To Implement EXZACT in Wheat
Transient Assays
Transformation System
Throughput High Low
Ability to regenerate plant
Low High
Comparison of steps
• Sequence AHAS genes• Design ZFNsZFN Design
• Assess ZFN efficacy • Optimise editing efficacyTransient Assays
• Generate wheat plants with precise genome modifications
Transformation System
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Nucleotide variation between duplicated copies of AHAS gene in wheat genome.
AHAS Sequencing For ZFN Design
TCGCCCAAACCCTCGCCGCCGCCATGGCCGC[a/c]GCCACCTCCCCCGCCGTCGCATTCTCGGGCGCC[gccgccg/a]CCGCCGCCGCCAT[a/g]CCCAAACCCGCCCGCCA[g/t]CCTCTCCCGCGCCACCAGCCCG[c/t]CTCGCGCCGCGCGCTCCCCGCCCGC[a/g]TCGTCAGGTG[c/t]TGCGCCGCGTCCCCCGCCGCCACCTCCG[t/c]CGCGCC[t/c]CCCGC[c/a]ACCGCGCTCCGGCC[a/g/c]TGGGGCCC[c/g]TCCGAGCCCCGCAAGGGCGCCGACATCCTCGTCGAGGCGCT[g/c]GAGCGCTGCGGCATCGTCGACGT[c/a]TTCGCCTACCC[t/c]GGCGGCGC[g/c]TCCATGGAGATCCACCAGGCGCTGACGCGCTCGCC[a/c]GTCATCACCAACCACCTCTTCCGCCACGAGCAGGGGGAGGCGTTCGCGGCGTCCGG[g/c]TACGCCCGCGCGTCCGGCCGCGTCGGCGTCTGCGTCGCCACCTCCGGCCCGGGGGCCACCAACCTCGTCTCCGCGCTCGC[c/t]GACGC[t/c]CTCCTCGACTCCATCCCCATGGTCGCCATCACGGGCCAGGTCCCCCGCCGCATGATCGGCACGGA[t/c]GCGTTCCAGGAGACGCCCAT[a/c]GTGGAGGTCACGCGCTCCATCACCAAGCACAACTACCTGGTCCTTGACGTGGAGGATATCCCCCGCGTCATCCAGGAAGCCTTCTTCCT[c/t]GC[a/g]TCCTCTGGCCGCCCGGGGCCGGTGCT[g/a]GTTGATATCCCCAAGGA[c/t]ATCCAGCAGCAGATGGC[t/c]GTGCC[t/c][g/a]TCTGGGACAC[g/t]CC[g/a]ATGAGTTTGCCAGGGTACATCGCCCGCCTGCCCAAGCCACCATCTACTGAATCGCTTGAGCAGGTCCTGCGTCTGGTTGGCGAGTCACGGCGCCCAATTCTGTATGTTGGTGGTGGCTGCGCTGC[a/g]TC[t/c]GG[t/c]GAGGAGTTGCGCCGCTTTGTTGAGCT[c/t]ACTGGGATTCC[a/g]GTTACAACTACTCT[t/g]ATGGGCCTTGGCAACTTCCCCAG[c/t]GACGACCCACTGTCTCTGCGCATGCT[t/g]GGGATGCATGGCACTGTGTATGCAAATTATGCAGT[c/a]GATAAGGCTGACCTGTTGCT[c/t]GCATTTGGTGTGCGGTTTGATGATCG[t/c]GTGAC[t/c]GGGAAAATCGAGGC[t/c]TTTGCAAGCAGGTCCAAGATTGAGCACATTGACATTGACCCAGCTGAGATTGGCAGAACAAGCAGCCACATGTCTCCATTTGTGCAGATGTTAAGCTTGCTTTACAGGGGTTGAATG[a/c]TCTATTAAATGGGAGCAAAGCACAACAGGGTCTGGATTTTGGTCCATGGCACAAGGAGTTGGATCAGCAGAAGAGGGAGTTTCCTCTAGGATTCAAGACTTTTGG[c/t]GAGGCCATCCCGCCGCAATATGCTATCCAGGTACTGGATGAGCTGACAAAAGGGGAGGCGATCATTGC[c/t]AC[t/c]GGTGTTGGGCAGCA[c/t]CAGATGTGGGCGGCTCAGTATTACACTTACAAGCGGCCACGGCAGTGGCTGTCTTC[a/g]TC[t/c]GGTTTGGG[t/g]GCAATGGGATTTGGGTT[a/g]CCAGCTGCAGCTGGCGCTGCTGTGGCCAACCCAGGTGTTACAGTTGTTGACATTGATGG[a/g/t]GATGGTAGTTTCCTCATGAACATTCAGGAGTTGGC[a/g]TTGATCCG[c/t]ATTGAGAACCTCCC[a/t]GTGAAGGTGATGATATTGAACAACCAGCATCTGGGAATGGTGGTGCA[a/g]TGGGAGGATAGGTTTTACAAGGCCAA[t/c]CGGGCGCACACATACCTTGGCAACCCAGAAAATGAGAGTGAGATATATCCAGATTTTGTGACGATTGCTAAAGGATTCAACGTTCC[a/g]GCAGTTCG[a/t]GTGACGAAGAAGAGCGAAGTCACTGCAGCAATCAAGAAGATGCTTGAGACCCCAGGGCCATACTTGTTGGATATCAT[a/t/c]GTCCCGCATCAGGAGCACGTGCTGCCTATGATCCCAAGCGGTGGTGCTTT[c/t]AAGGACATGATCATGGAGGGTGATGGCAGGACCTCGTACTGAAATTTCGACCTACAAGACCTACAAGTGTGACATGCGCAATCAGCATG[a/g]T[g/a]CC[c/t]GCGTGTTGTATCAACTACT[a/g]GGGGTTCAACTGTGA[a/g]CCATGCGTTTTCTAGTTTGCTTGTTTCATTCATATAAGCTTGT[a/g]TTACTTAGTTCCGAACC[c/g]TGTAG[c/t]TTTGTAGTCT[a/c]TG[c/t]TCTCTTTTGTAGGGATGTGCTGTCATAAGAT[a/g]TCATGCAAGTTTCTTGTCCTACATATC
ZFNs designed to cut AHAS wheat gene in one or more sub-genomes
High ZFN design potential for narrow target sequence window
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Transient assays used to assess ZFN cleavage efficacy and specificity.
ZFN Cleavage Efficacy and Specificity
ZFNs cut wheat genome in sub-genome and target gene-specific manner
A-genome B-genome D-genome Specificity
pDAB109354 54,781 (1.0-fold) 75,916 (1.3-fold) 73,067 (1.3-fold) Similar cleavage efficiency for all sub-genomespDAB109358 607 (1.0-fold) 268 (3.7-fold) 34,852 (130-fold) D-genome specific cleavage
Deletion size (bp)
Fre
quen
cy (
%)
ZFN activity (ppm) = No. reads with NHEJ deletion x 10^6 No. wild-type reads
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Transient assays demonstrate ZFN-mediated gene knock-out and excision.
Transfection with single ZFN revealed evidence for efficient gene knock-out (loss-of-function), while co-transfection with two ZFNs showed efficient gene excision, evidenced by prevalence of AHAS-derived reads lacking intervening sequence between ZFN cut sites.
Strategies For EXZACT Delete & Excise
Molecular evidence generated for EXZACT Delete and Excise modes
Delete: Gene knock-out(using single ZFN)
Excise: Gene excision(using paired ZFNs)
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Transient assays used to investigate approaches for ZFN-mediated gene addition and gene editing via NHEJ- and HR-directed DNA repair pathways.
Strategies For EXZACT Add & Edit
Editing frequency (ppm) = No. reads with edits x 10^6 No. wild-type reads
NHEJ-directed DNA repair HR-directed DNA repair
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Strategies For EXZACT Add
Number edits per million reads
Example of transient assay showing molecular evidence for EXZACT Add via NHEJ-directed DNA repair pathway.
Transfected with ZFN designed to cleave all AHAS gene copies and a linear double-stranded DNA donor
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Strategies For EXZACT Edit
Number edits per million reads
Example of transient assay showing molecular evidence for EXZACT Edit via HR-directed DNA repair pathway.
Transfected scutella with ZFN designed to cleave all AHAS gene copies and plasmid DNA donor
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Molecular evidence generated for EXZACT Add and Edit modes based on NHEJ- and HR-directed DNA repair
Strategies For EXZACT Add & Edit
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Recovered AHAS edited wheat plants tolerant to Imazamox
Transformation system based on biolistics-mediated DNA delivery to scutella of immature zygotic wheat embyros.
Target mutation: S653N → Confers tolerance to Imidazolinone class herbicides
Generation of AHAS Edited Wheat Plants
DNA delivery comprised a ZFN-expressing plasmid construct (designed to cleave all AHAS gene copies) and a double-stranded linear donor (designed to utilise NHEJ-directed DNA repair pathway). AHAS edited plants were recovered using Imazamox selection.
Donor designed to maintain correct protein coding after integration at site of DSB
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Sanger sequence characterisation of T0 AHAS edited events.
Generation of AHAS Edited Wheat Plants
Allele 1 Allele 2 Allele 1 Allele 2 Allele 1 Allele 2Event 10 Perfect Edit Unedited Unedited n.d. Unedited n.d.
Status Functional S653N mutation . . . . .No. sequences* 10 19 37 0 29 0
Event 6 Unedited n.d. Perfect Edit Unedited Unedited n.d.Status . . Functional S653N mutation . . .
No. sequences 22 0 11 18 43 0Event 11 Unedited n.d. Unedited n.d. Perfect Edit Unedited
Status . . . . Functional S653N mutation .No. sequences 35 0 37 0 15 11
Event 9 Perfect Edit n.d. Imperfect Edit Unedited Unedited n.d.Status Functional S653N mutation . Non-sense frameshift . . .
No. sequences 24 0 13 21 33 0Event 1 Perfect Edit NHEJ deletion at ZFN Imperfect Edit Unedited Imperfect Edit Unedited
Status Functional S653N mutation Non-sense frameshift Non-sense frameshift . Non-sense frameshift .No. sequences 13 20 12 19 14 22
Event 4 Perfect Edit Unedited Imperfect Edit Unedited Perfect Edit Imperfect EditStatus Functional S653N mutation . Non-sense frameshift . Functional S653N mutation Non-sense frameshift
No. sequences 6 11 44 30 6 11
A-genome B-genome D-genome
*indicates number of independent PCR amplicon clones sequenced.
Of recovered events, 46% had single gene edits; remainder had editing ≥2 AHAS genes Stable Mendelian inheritance of AHAS edits and Imazamox herbicide tolerance
phenotype was confirmed in T1 and T2 generations
Representative AHAS editing outcomes in independent events.
18DEPARTMENT OF ECONOMIC DEVELOPMENT,
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Generated Imazamox tolerant wheat plants via EXZACT Add, with independent plants demonstrating
single perfect AHAS allele edits on A-genome, B-genome and D-genome
simultaneous AHAS editing in multiple sub-genomes
hemizygous and homozygous sub-genome-specific AHAS allele editing
Generation of AHAS Edited Wheat Plants
Perfect AHAS allele-splicing in A-genome
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Imperfect EXZACT Add outcomes typically had small deletions at the left- or right-border of the integrated donor.
Generation of AHAS Edited Wheat Plants
Imperfect AHAS allele-splicing in B-genome
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Extending the platform technology to generate wheat plants with precise genome modifications at non-selectable trait loci.
e.g. Gene editing at an endogenous trait locus for grain quality (non-selectable) by simultaneous co-editing at a second (selectable) locus.
EXZACT Modification of Non-Selectable Loci
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Demonstrating EXZACT co-editing using AHAS as a non-selectable trait locus.
EXZACT Modification of Non-Selectable Loci
AHAS(S653)−
AHAS(N653)PAT
AHAS(N653)PAT
AHAS(N653)−
Imazamox® Susceptible Tolerant Tolerant Tolerant
Basta® Susceptible Tolerant Tolerant Susceptible
Non-Selectable LocusSelectable Locus
EXZACT co-editing simultaneously introduces precise AHAS edits and integrates selectable marker PAT at an independent locus. Wheat events are recovered using BASTA® selection. Wheat plants with precise AHAS edits and without exogenous selectable marker are recovered by screening segregating T1 plants.
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Demonstrating EXZACT co-editing using AHAS as a non-selectable trait locus.
Target mutation: S653N → Confers tolerance to Imidazolinone class herbicides
EXZACT Modification of Non-Selectable Loci
DNA co-delivered:• ZFN-expressing plasmid
construct designed to cleave AHAS genes
• Double-stranded linear donor designed to utilise NHEJ-directed DNA repair pathway.
• PAT-expressing plasmid construct
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• Implementation of EXZACT technology in wheat was achieved.
• World’s first EXZACT wheat plants were generated.
• Transient assay systems for generating molecular evidence for precision editing in wheat were used to demonstrate strategies for EXZACT implementation of Delete, Edit and Add modes using HR- and NHEJ-directed DNA repair.
• Multiple options exist for generating wheat plants with precise integrative and non-integrative editing at both selectable and non-selectable trait loci.
• EXZACT technology enables the efficient generation of wheat plants with
precise sub-genome-specific (or target gene-specific) edits simultaneous precise editing in multiple sub-genomes (or gene targets)
through ZFN design and/or donor construct design.
• A transformation system for routine generation of selectable marker-free wheat plants with precise genome modifications at non-selectable trait loci was established.
Summary
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• For wheat crop improvement, EXZACT technology enables:
More efficient transgene screening – precision transformation at a pre-defined locus should reduce event-to-event variability, compared to random integration events, thereby allowing more genes and constructs designs to be tested with the same resource base.
Rapid generation of targeted mutations and loss-of-function alleles for conventional forward breeding – expected to be faster than other types of mutational breeding, e.g. TILLING.
Creation and use of new alleles for trait improvement.
Summary
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Acknowledgements
DEPI
Yidong RanNicola PatronMargaret BuchananYi TiYu Hua WangYingying CaoQuoqing TaoYihan LinHeather AndersonJohn MasonPippa KayDebbie WongJoanna PetkowskiKaterina VikudaSami HakimLinh NguyenGerman Spangenberg
Dow AgroSciences
Terry WalshMike AinleySteve Webb
Project Funding Source