Genome Engineering and Haploid Technology in Crop Plants

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1 Genome Engineering and Haploid Technology in Crop Plants Jochen Kumlehn Plant Reproductive Biology Leibniz Institute of Plant Genetics and Crop Plant Research (IPK) Gatersleben

2 Genetic modifications in plant domestication und breeding Spontaneous mutagenesis Intraspecific recombination (sexual reproduction)

3 Genetic modifications in plant domestication und breeding Spontaneous mutagenesis Intraspecific recombination (sexual reproduction) Interspecific recombination

4 Genetic modifications in plant domestication und breeding Spontaneous mutagenesis Intraspecific recombination (sexual reproduction) Interspecific recombination Induced mutagenesis A.M. Stanca

5 Genetic modifications in plant domestication und breeding Spontaneous mutagenesis Intraspecific recombination (sexual reproduction) Interspecific recombination Induced mutagenesis Genetic transformation

6 Genome engineering site directed genome modification using customizable endonucleases Meganucleases TAGGGATAACAGGGTAAT ATCCCTATTGTCCCATTA N RVDs: left TALEN unit TALE Nucleases FokI C TCGAACAAGACCGTGTAgggataacagggta ccctattgtcccattaaggctccagtcgaat NG HD NI NN C FokI spacer bp right TALEN unit N Zincfinger Nucleases (ZFNs) left ZFN unit N FokI C ACCGTGTAGGGAtaacag attgtcccattaaggctc zinc fingers: C FokI N right ZFN unit RNA guided Nucleases Cas9 protospacer (20 bp) PAM agaccgtgtagggataacagggtaatatggtccgtcg tctggcacatccctattgtcccattataccaggcagc GUAGGGAUAACAGGGUAAUA sgrna TALE CAS CRISPR Transcription Activator Like Effector CRISPR Associated Clustered Regularly Interspaced Short Palindromic Repeats

7 Transcription activator like (TAL) effectors of Xanthomonas species DNA binding domain N Activation C Domain C A T C G C C A A G C T G G C A C T G T A G C G G T T C G A C C G T G A T A G C G G T C C T A T.. A T C G C C A G G A T A.. Activation of plant gene expression Xanthomonas host plant cell Pol II mod. after Nino Liu et al. 2006

8 Transcription activator like (TAL) effectors of Xanthomonas species DNA binding domain N C C A T C G C C A A G C T G G C A C T G T A G C G G T T C G A C C G T G A T A G C G G T C C T A T.. A T C G C C A G G A T A.. Four types of repeats: Specifically bound nucleotides: A T G C Boch et al., Science 2009 DNA binding domains can be designed for any DNA target of choice. Their coding seqs can be generated and expressed in any organism.

9 Site directed mutagenesis using customized endonucleases FokI...c t t a c c t C A T C G C C A A G C T G G C A C C c t t g t t c a a g c g G A C A G C A A T A C C G A A T g g a a g t g......g a a t g g a G T A G C G G T T C G A C C G T G G G A a c a a g t t c g c C T G T C G T T A T G G C T T A c c t t c a c... plant gene of interest FokI Double strand break...c t t a c c t C A T C G C C A A G C T G G C A C C c t t g t t c a a g c g G A C A G C A A T A C C G A A T g g a a g t g......g a a t g g a G T A G C G G T T C G A C C G T G G G A a c a a g t t c g c C T G T C G T T A T G G C T T A c c t t c a c... DSB Repair via non homologous end joining error free...c t t a c c t C A T C G C C A A G C T G G C A C C c t t g t t c a a g c g G A C A G C A A T A C C G A A T g g a a g t g......g a a t g g a G T A G C G G T T C G A C C G T G G g a a c a a g t t c g c C T G T C G T T A T G G C T T A c c t t c a c... error prone...c t t a c c t C A T C G C C A A G C T G G C A C C c t t g t t - a a g c g G A C A G C A A T A C C G A A T g g a a g t g......g a a t g g a G T A G C G G T T C G A C C G T G G g a a c a a - t t c g c C T G T C G T T A T G G C T T A c c t t c a c... Any genomic sequence of choice can be mutated in planta.

10 Site directed mutagenesis by TALEN expression in haploid cells Agrobacterium with binary for left TALEN unit Agrobacterium with binary for right TALEN unit Daghma et al. (2014) Frontiers in Plant Science Gurushidze et al. (2014) PLoS One co transfer of TALEN units to embryogenic pollen of GFP transgenic barley GFP used as target gene DIC CLSM

11 Site directed mutagenesis by TALEN expression in haploid cells Gurushidze et al. (2014) PLoS One Identification of GFP knock out plants white light Fluorescence GFP transgenic line (positive control) GFP transgenic line mutated by means of GFP specific TALENs

12 Site directed mutagenesis by TALEN expression in haploid cells Gurushidze et al. (2014) PLoS One N RVDs: TALE nucleases TCGAACAAGACCGTGTAgggataacagggta ccctattgtcccattaaggctccagtcgaat NG HD left TALEN unit NI NN C FokI C FokI spacer bp right TALEN unit N target specific mutations in ca. 20% of TALEN transgenic plants WT gene left TALEN binding site right TALEN binding site TCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCT 24/2 1 TCGAGGGCGAC TTCAAGGAGGACGGCAACATCCT 36 1n 2n 21 T1 no segregation 24/2 2 TCGAGGGCGACACCCTGGTG CCCCTGGTGTACTTCAAGGAGGACGGCAACATCCT 25 2n T1 no segregation 24/2 9 TCGAGGGCGAC TTCAAGGAGGACGGCAACATCCT 36 24/3 4 TCGAGGGCGACACCCTGGTGAACCGCATCGAG CGACTTCAAGGAGGACGGCAACATCCT 11 fs 32/2 2a TCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGG CGACTTCAAGGAGGACGGCAACATCCT 04 2n fs 33 T1 segregation Haploid technology allows for the production of instantly homozygous mutant plants.

13 Site directed mutagenesis in barley using TALENs Maia Gurushidze, Ingrid Otto, Götz Hensel, Hannes Trautwein Agrobacterium with left GFP specific TALEN unit Agrobacterium with right GFP specific TALEN unit Retransformation of GFP transgenic line with left GFP TALEN Selection of homozygotes Retransformation of GFP transgenics line with right GFP TALEN Selection of homozygotes > 200 F1 hybrid plants (GFP + both TALENs) > 20 analysed in detail GFP amplicons subcloned and sequenced RT PCR for FOKI RT PCR for FOKI 4 8 different mutations per plant, GFP WT only rarely detected 4 lines X 7 lines

14 Mutations of 8 TALEN hybrid plants GFP-WT left TALEN binding site right TALEN binding site AGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATA Hannes Trautwein, Maia Gurushidze 48/1a 49/3a 49/3b 43/3a 56/1a 48/1b 49/3c 53/3a 39/4a 49/3e 49/3f 49/3g 49/3h 53/3b 53/3c 53/3d 53/3e 53/3f 7/1a 56/1b 7/1c 7/1d 56/1c 58/4a 58/4b 58/4c 58/4d 39/4c 39/4d 39/4e 39/4f 39/4g 56/1d 39/4h 39/4i 56/1e 56/1f 56/1g 43/3b 43/3c 48/1c 48/1d 43/3d 48/1e 58/4e AGTTCGAGGGCGACACCCTGGTGAACCGCATCGA CTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATA AGTTCGAGGGCGACACCCTGGTGAACCGCATCGA CTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATA AGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGG CAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATA AGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGG----CGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATA AGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGG----CGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATA AGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGG----CGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATA AGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGG----CGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATA AGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGG----CGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATA AGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGG----CGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATA AGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGG----GGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATA AGTTCGAGGGCGACACCCTGGTGAACCGCATCGAG CTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATA AGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGG AGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATA AGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGG-CATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATA AGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTG AGTACAACTACAACAGCCACAACGTCTATA AGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGG--TCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATA AGTTCGAGGGCGACACCCTGGTGAACCGCATCGA CTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCAGGAGTACAACTACAACAGCCACAACGTCTATA AGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAA------CGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATA AGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAG GAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATA AGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAG GAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATA AGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAG GAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATA AGTTC TGAGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATA AGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAG ACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATA AGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAG ACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATA AGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGG---CGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATA AGTTCGAGGGCGACACCCTGGTGA CTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATA AGTTCGAGGGCGAC TTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATA AGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGG TGTACAACTACAACAGCCACAACGTCTATA AGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGA CTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATA AGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAG CTTCAAGGAGGACGGCAACATCCTGGGGCACAGGCTGGAGTACAACTACAACAGCCACAACGTCTATA AGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGG---TCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATA AGTTCGAGGGCGACACCCTGG CGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATA AGTTCGAGGGCGACACCCTGGTGAACCACATCGAGCTGAAGG CACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATA AGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGG CACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATA AGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAG TACAACTACAACAGCCACAACGTCTATA AGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAG TACAACTACAAGGAGTACAACTACAACAGCCACAACGTCTATA AGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGG GGACACCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATA AGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGG--TCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATA AGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGG---AGGACTTCAAGGAG TACAACTACAACAGCCACAACGTCTATA AGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGG---AGGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATA AGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGC CGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATA AGTTCGAGGGCGACACCCTGGTGAACCG CGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATA AGTTCGAGGGCGACACCCTGGTGAACCGCATCGA ACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATA AGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGTACTGGGGCACAAGGAGGACGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAG AGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCAACATCCTGGGCGACTCCTTCAGCTGGAGTACAACTACAACATCCTGGGGCACAAGCTGGAGTACAACTACAA AGTTCAAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGTACAACTACATGTATACCTATCCTAGATCGATATTTCCATCCATCTTAAACTCGTAACTTCAAGGAGGACGGCAAC / / / / /+2-5/ /+19-19/+36-8/+57

15 Inheritance of mutations and chimera dissolution Maia Gurushidze, Hannes Trautwein primary mutants are typically heterozygous and chimeric only those mutations present in gametophytes (embryo sac, pollen) are heritable via selfing, homozygous mutants occur only rarely DH progeny of one primary mutant left TALEN binding site right TALEN binding site WT gene GAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGG DH61/1a DH61/1b DH61/1c DH61/1d DH61/1e DH61/1f DH61/1g DH61/1h DH61/1i DH61/1j GAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGA CTTCAAGGAGGACGGCAACATCCTGGGG GAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGG GAGGTGAA CTTCAAGGAGGACGGCAACATCCTGGGG GAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGG GAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGG GAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGG GAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGG GAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGA CTTCAAGGAGGACGGCAACATCCTGGGG GAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGA-----CTTCAAGGAGGACGGCAACATCCTGGGG GAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCAACTGAGAAAGATGAGCTATATCATCTTTCTCAGACTTGCGATGATTA -15 WT -48 WT WT WT WT /+75 Most mutations detected in primary mutants proved heritable. Various alleles of primary mutants can be readily separated and fixed in DH plants.

16 Homology directed genome editing DSB...c t t a c c t C A T C G C C A A G C T G G C A C C c t t g...g a a t g g a G T A G C G G T T C G A C C G T G G G A a c a g c g G A C A G C A A T A C C G A A T G G a a g t g... t c g c C T G T C G T T A T G G C T T A C C t t c a c... DSB repair via homologous recombination...c t t a c c t C A T C G C C A A G C T G G C A C C c t t g...g a a t g g a G T A G C G G T T C G A C C G T G G G A a c G A G A c t c t a g c g G A C A G C A A T A C C G A A T G G a a g t g... t c g c C T G T C G T T A T G G C T T A C C t t c a c......c t t a c c t C A T C G C C A A G C T G G C A C C c t t g G A G A a g c g G A C A G C A A T A C C G A A T G G a a g t g......g a a t g g a G T A G C G G T T C G A C C G T G G g a a c c t c t t c g c C T G T C G T T A T G G C T T A C C t t c a c... recombinant DNA repair template including modification of choice Options predefined insertions, deletions allele exchange precise editing of single nucleotides and amino acids

17 Homology directed genome editing in barley Nagu Budhagatapalli, Sindy Schedel, Stefan Hiekel, Götz Hensel GFP transgenic cell ZmUbiP GFP TALEN left T 2 3 % of viable TALEN transgenic cells show gfp to yfp conversion ZmUbiP GFP TALEN right T controls: no gfp to yfp conversion when any one of the 3 vectors was omitted DNA repair template N truncated YFP T/Y precise editing of gdna confirmed by sequencing GFP filter set merged YFP filter set

18 RNA guided Cas9 endonuclease standard Cas9 derives from Streptococcus pyogenes target gdna protospacer (20 bp) PAM agaccgtgtagggataacagggtaatanggtccgtcg tctggcacatccctattgtcccattatnccaggcagc GUAGGGAUAACAGGGUAAUA guide RNA Cas9

19 RNA guided Cas9 endonuclease target gdna protospacer (20 bp) agaccgtgtagggataacagggtaatanggtccgtcg tctggcacatccctattgtcccattatnccaggcagc PAM GUAGGGAUAACAGGGUAAUA Cas9 guide RNA

20 Transient expression test for customized endonuclease constructs Budhagatapalli et al. (2016) Plant Methods Microparticle bombarded barley cell ZmUbiP ER SP target seq restored GFP fs XFP KDEL T (every 3rd indel) indel ZmUbiP mcherry T mcherry detection ZmUbiP Cas9 T OsU3P grna T GFP detection

21 Transient expression test for customized endonuclease constructs Transient co expression in barley leaf epidermis of: P ZmUbi1 ::Cas9 P OsU3 :grna (as specified below) test vector carrying target seq upstream of out of frame YFP P ZmUbi1 ::mcherry Budhagatapalli et al. (2016) Plant Methods YFP/mCherry ratio [%] GFP grna1 GFP grna2 HvCENH3α grna3 HvCENH3α grna5 HvCENH3α grna13 HvCENH3β grna13 HvCENH3β grna25

22 grna/cas9 mediated mutagenesis of barley CenH3α >100 T 0 plants (positive for grna and Cas9) Stefan Hiekel cenh3 mutations detected in ca. 20% 35 independent mutations 17 different mutations only in frame mutations inherited (CenH3α is essential for gamete functionality) number of independent mutation events insertion deletion combined ± size of mutation [bp]

23 Summary Gurushidze et al. (2014) PLoS One Budhagatapalli et al. (2015) G3: Genes Genomes Genetics Budhagatapalli et al. (2016) Plant Methods Site directed mutagenesis in barley using TALENs can be highly efficient. Current TALEN technology is still too unreliable for routine use in barley. Chimera dissolution and mutant fixation is greatly facilitated by haploid technology. A test system for customizable endonucleases was established. Precise genome editing (conversion of gfp into yfp) was exemplified at the barley cell level. RNA guided Cas9 routinely works in barley.

24 IPK Gatersleben, Plant Reproductive Biology Postdocs Nagu Budhagatapalli Götz Hensel PhD students Stefan Hiekel Christian Hertig Krishna Mohan Pathi Sindy Schedel Technical assistants Carola Bollmann Heike Büchner Theresa Engling Petra Hoffmeister Conny Marthe Ingrid Otto Sabine Sommerfeld Undergrads Jan Dirks Pascal Ganz Kaysar Kaiyoum Cooperators at IPK A. Houben, T. Ishii M. Melzer, T. Rutten N. Stein, P. Schweizer External cooperators K. Schmidt, M. Nießen (KWS) A. Hanemann (Breun Saatzucht) S. Kusch, R. Panstruga, (RWTH Aachen) R. Morbitzer, T. Lahaye (Univ. Tübingen) Funding BMBF BMEL DFG ERA CAPS Former members Diaa Daghma Sibylle Freist Maia Gurushidze Andrea Müller Stefanie Pencs Anne Kathrin Pfrieme