Gene editing in cereals

Transcription

1 University of Minnesota Gene editing in cereals Mick Ayliffe

2 The importance of cereals in Australian agriculture VALUE OF AGRICULTURAL COMMODITIES PRODUCED IN Australia (year ended 30 June 2016)

3 Gene editing in cereals wheat Platforms Oligonucleotide directed targeting Mega nucleases Zn 2+ finger nucleases TALENs CRISPR/Cas9 9bp FokI nuclease FokI nuclease 9bp barley, rice

4 TALEN editing in wheat Wheat Origin Transf. eff Stable wheat transgenics produced using high efficiency Fielder USA 41% Japan Tobacco Agrobacterium system Gladius Aust 32% Westonia Aust 45% Mace Aust 25% Stewart - durum Can 26% Kronos - durum USA 51% Mutus Mexico 24% Kachu Mexico 40% Navajoa Mexico 27%

5 TALEN editing in wheat TALEN targeting of a pre-existing transgene in wheat LB Ubi TALEN GUS1 nos NPTII RB LB Ubi TALEN GUS2 nos NPTII RB co-transformation LB Ubi uida nos BAR RB Fielder wheat line containing single GUS transgene T0 lines selected with all three transgenes

6 TALEN editing in wheat TALEN deletions observed in the GUS gene uida PCR RE assay 18c 17b 21b 2a 32a ve BclI TALEN GUS1 TALEN GUS2 GUS targetgtgggcattcagtctggatcgcgaaaactgtggaattgatcagcgttggtgggaaagcgcgttacaagaaagccgggcaatt Plant 45 GTGGGCATTCAGTCTGGATCGCGAAAACTGTGGAATT----AGCGTTGGTGGGAAAGCGCGTTACAAGAAAGCCGGGCAATT Plant 21B GTGGGCATTCAGTCTGGATCGCGAAAACTGTGGAATT---CAGCGTTGGTGGGAAAGCGCGTTACAAGAAAGCCGGGCAATT Plant 38 GTGGGCATTCAGTCTGGATCGCGAAAA CAGCGTTGGTGGGAAAGCGCGTTACAAGAAAGCCGGGCAATT digestion resistant products cloned and sequenced 25% of T0 wheat showed cleavage of GUS (3/12 transgenics)

7 TALEN editing in wheat Gene editing in the absence of a repair template ie. (NHEJ) usually causes gene knockout (excluding promoter sequences) But these events, unlike repair template events, are the most likely to be considered as nongm (if any)????! Can NHEJ be used to recreate a gene function?? Can we bring a pseudogene back from the dead!!!

8 TALEN editing in wheat wt Lr21 rust resistance gene provides resistance to wheat leaf rust Lr21 Fielder lr21 psuedogene lr21 1 bp deln in ORF = out of frame Can we restore ORF frame and function by NHEJ? - Deletion occurs in nonconserved region of the protein - 5 regulatory region shown to be functional (Huang et al, 2009) - protein differs by just two a.a. compared with wt

9 TALEN editing in wheat Fielder co-transformed with a Lr21 TALEN pair LB Ubi TALEN Lr21a nos NPTII RB LB Ubi TALEN Lr21b nos NPTII RB Fielder Lr21 psuedogene TALEN Lr21a TALEN Lr21b cacgtttgcaggatatattcaagattacataa-ggaggaatgcaaggatgagaaacttttcgaca Homozygous 3 bp deletion Numbers = no. T0 plants with alteration at this site T0 Fielder wheat plants CAAGATTACATAA-GGAGGAATGCAAGGATGAGAAACTTTTCGACA 28 plants (80%) had target site alteration T0 plant is homozygous for a 4bp deletion

10 TALEN editing in wheat Heritability of gene modification Mendelian in some cases with no evidence of T0 chimerism Plant 14 Head 1 Head 2 Head 3 Head 4 Total wt het mt new? X 2 3:1 p = 0.62 Plant 27 No inheritance of a mutant allele amongst 38 T1 screened

11 TALEN editing in wheat T1 progeny homozygous for modified alleles Lr21 P14 P20 P20 P16 P24 P24 tttgcaggatatattcaagattacataaaggaggaatgcaaggatgagaaactt Tttgcaggatatattcaagattac------aaggaatgcaaggatgagaaactt tttgcaggatatattcaagattac------aaggaatgcaaggatgagaaactt tttgcaggatatattcaaga ggaatgcaaggatgagaaactt tttgcaggatatattcaagatta atgcaaggatgagaaactt tttgcaggatatattcaagattac aggaatgcaaggatgagaaactt tttgcaggatatattcaagattaca-----gaggaatgcaaggatgagaaactt in frame stop codon out of frame Proteins encoded by restored alleles homozygous in T1 progeny Lr21 P14 P20 P20 YSVIGIYGVAGSGKTTFAGYIQDYIKEECKDEKLFDTIMCIHVTETFSVDDIF YSVIGIYGVAGSGKTTFAGYIQDY-K-ECKDEKLFDTIMCIHVTETFSVDDIF YSVIGIYGVAGSGKTTFAGYIQDY-K-ECKDEKLFDTIMCIHVTETFSVDDIF YSVIGIYGVAGSGKTTFAGYIQ----EECKDEKLFDTIMCIHVTETFSVDDIF

12 TALEN editing in wheat Do any of these restored full length proteins provide rust resistance? Challenged T1 seedlings with wheat leaf rust Lr21 lr21

13 TALEN editing in wheat Do any of these restored full length proteins provide rust resistance? NO! Moral of the story - TALENs work with high efficiency in wheat - it is easier to destroy gene function than create even with gene editing!!

14 CRISPR/Cas9 editing in barley Brett Ford Simultaneous gene editing of a barley gene family Barley Flower Promoting Factor 1-like gene family targeted with CRISPR FPF1-like FPF1-like Two genes on 1 chromosome FPF1-like Third gene on another chromosome Single grna target U6 grna Ubi pcas9 Tnos

15 CRISPR/Cas9 editing in barley Brett Ford 12 transgenics made in barley cultivar Golden Promise 6 T0 showed evidence of CRISPR activity (50%) 2 (18%) had all 3 genes cleaved although monoallelic FPF1-likeA Ref Morex seq Golden Promise BF10-2 BF10-3 The 2 lines below have mutations in all 3 targeted FPF1-like genes wt mutant FPF1-likeB FPF1-likeD Ref Morex seq Golden Promise BF10-2 BF10-3 Ref Morex seq Golden Promise BF10-2 BF10-3 wt mutant wt mutant

16 CRISPR/Cas mediated gene editing in rice Ming Luo Multiplex gene editing of a tightly linked rice gene pair 10kb OsFIE1 OsFIE2 T1 T2 T3 T2 Targeted two tightly linked endosperm developmental genes OsFIE represses endosperm development U6 T1 grna U6 T2 grna U6 T3 grna Three target sites Ubi pcas9 Tnos Three different grnas

17 CRISPR/Cas mediated gene editing in rice Ming Luo OsFIE1: Mutations of OsFIE1andOsFIE2 at T1 WT(T1): TGGTGGAGTAGGAGGTCACCGACACGAAGTACTTGGTGTTGTAAGTTGCTTAAGCT Homo1: TGGTGGAGTAAGAGGTC...GGTGTTGTAAGTTGCTTAAGCT -17 Heter1: TGGTGGAGTAGGAGGTCACCGACACGAAGTAACTTGGTGTTGTAAGTTGCTTAAGCT +1 Bi-allelic: TGGTGGAGTAGGAGGTCACCGACACGA...ACTTGGTGTTGTAAGTTGCTTAAGCT -3 TGGTGGAGTAGGAGGTCACCGACACGAAGTTACTTGGTGTTGTAAGTTGCTTAAGCT +1 WT(T2): TCAACCATCGAATCCTTCACTCATCATTTCTGCAAGCAAGGTTAGAAGATTACAT Hete2: TCAACCATCGAATCCTTCACTCATCATTTCTGTAAGCAAGGTTAGAAGATTACAT +1 OsFIE2: WT(T2): ATAAGAACTCAAGCATTAAAGCCTTCGCTCATCATTTCTGCAAGCAAGGTTTGAAGA Heter1: ATAAGAACTCAAGCATTAAAGCCTTCGCTCATCATTTCTGC..GCAAGGTTTGAAGA -2 Heter2: ATAAGAACTCAAGCATTAAAGCCTTCGCTCATCATTTCT...GCAAGGTTTGAAGA -4 Heter3: ATAAGAACTCAAGCATTAAAGCCTTCGCTCATCATTTC...GCAAGGTTTGAAGA -5 Heter4: ATAAGAACTCAAGCATTAAAGCCTTCGCTCATCATTTCTG...GCAAGGTTTGAAGA -3 50% T0 rice had at least one allele edited Homozygous and biallelic KOs identified in T0 generation 4/100 T0 plants had both linked genes mutated Heter & Bi-allelic ATAAGAACTCAAGCATTAAAGCCTTCGCTCATCATTTCTGCAAGCAAGGTTTGAAGA ATAAGAACTCAAGCATTAAAGCCTTCGCTCATCATTTCT...GCAAGGTTTGAAGA -4 ATAAGAACTCAAGCATTAAAGCCTTCGCTCATCATTTCTGC..GCAAGGTTTGAAGA -2

18 CRISPR/Cas mediated gene editing in rice Ming Luo 10kb OsFIE1 OsFIE2 WT A B C E F Mutant phenotypes observed in these four T0 plants ie. endosperm formation in the absence of fertilisation G J Mendelian pollen transmission of double KO haplotype in these 4 plants W T Very difficult to achieve co-mutation of such tightly linked genes by conventional mutagenesis fie1 fie2 ~50% unfertilised ovules had autonomous endosperm development + +

19 Gene editing in cereals TALENs and CRISPR/Cas are good tool for gene KOs in wheat, barley and rice Gene replacement and gene targeting is where we want to be in future

20 Gene editing in cereals Potential future application Gene stacking of rust disease resistance genes in wheat R gene1 R1 R2 R3 R4 R5 Single pathogen mutation 5 independent mutations needed Overcome short lived Durable resistance Inherited as single locus

21 Gene editing in cereals 44 kb Lb Rb Hy Sr46 Sr45 Lr67 Sr50 Sr35 Sr22 R S R gene stacks with up to 6 genes Transgenic wheat with all genes full length Genes inherited as a single locus Resistance segregates as a single locus

22 Gene editing in cereals Can we add and subtract to the stack using gene editing? SrX Sr46 Sr45 Lr67 Sr50 Sr35 Sr22 Real utility!

23 Gene editing in cereals Summary - Highly efficient TALEN editing in wheat of a transgene and an endogenous gene - Simultaneous editing of a dispersed barley gene family in T0 plants - Multiplex gene editing of a tightly linked rice gene pair

24 Acknowledgements CSIRO Ag & Food Ming Luo Brett Ford Amy Rinaldo Xiaoba Wu Soma Chakraborty Melanie Soliveres Terese Richardson Dhara Bhatt Smitha Louis University of Tuebingen Thomas LaHaye Robert Morbitzer John Innes Institute Brande Wulff Burkhard Steuernagel South China Agriculture University Yaoguang Liu