CRISPR/Cas9 Based Gene Editing in Barley & Potato: From Construct Design to Mutation

Screening

Abdellah Barakate

abdellah.barakate@hutton.ac.uk

Use of CRISPR/Cas9 tools in plants at the James Hutton Institute & Dundee University

Gene validation toolbox in plants

mRNA

Gene silencing

sense antisense amiRNA

mRNA Protein

Post-translation modifications

antisense miRNA

- TILLING - T-DNA & transposons - Gene targeting: CRISPR/Cas

Gene of interest transgene

Integration Candidate gene(s)

Validation

http://www.hutton.ac.uk/research/groups/cell-and-molecular-sciences

http://www.lifesci.dundee.ac.uk/research/ps

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The distribution of crossovers is skewed towards the ends of chromosomes

Barley chromosome sections. Keller & Krattinger (2017). Nature 544, 424–425.

DAPI

Distribution of crossovers in barley. Higgins et al. (2012). Plant Cell 10.1105/tpc.111.102483

Aims: - Mechanisms controlling COs? - Manipulation of COs.

High Throughput and Global analysis of Meiosis in Barley

Male meiotic cells in barley.

a

b c

d

PMC

c

Literature

Candidate gene(s)

Gene validation: - Gene editing

• CRISPR/Cas9 • TILLING

- Gene down-regulation (RNAi) - Gene over-expression with tags

Cytogenetics

- Genomics - RNA-seq - Proteomics - Epigenetics

Aniline blue staining DAPI staining

Chromosome organisation and recombination

Epigenetic marks

CRISPR/Cas9 construct design

1) T-DNA Plasmids

o pBract & pGreenII

(John Innes Centre, UK)

2) Cas9 (codon optimised)

o Human

o Barley (monocots)

o Tomato (dicots)

o NLS and promoters

3) gRNA

o Target: ontology, exons, polyploidy, polymorphism…etc.

o Spacer design: http://www.broadinstitute.org/rnai/public/analysis-tools/sgrna-design

o gRNA structure!

o Promoter: U6

4) Single-step cloning

5) Multiplexing gRNAs

o Multiple gRNA cassettes

o Single U6 promoter & tRNA

Explant bombardment Protoplasts (polyethyleneglycol )

Agrobacterium Transformation

(Jennifer Stephens, FUNGEN)

CRISPR/Cas9 based gene editing in Plants: screening methods

1) Screening of T0 lines for Indels (PCR based method)

- IDAA method

- Sanger sequencing

- Frameshift selection

- Miseq?

2) Screening of T1 lines for Cas9-free homozygotes

- Single Cas9 locus

- Multiplex-PCR and capillary DNA analyser.

- Sequencing

3) Phenotyping

PCR product sequencing

WT

Callus#2

Callus#15

Insertion?

Deletions?

Indel Detection by Amplicon Analysis (Capillary ABI3730 DNA analyser).

A summary of knockouts of barley meiotic genes

CRISPR/Cas9 constructs Transgenics T0 lines screening for Indels

Target genes Target sites no. Calli no. Plants Target gene(s) Indels distribution

figl1; fancm

FIGL1: 5'-end

35 62:

26 ST 29 NS

wt 32

figl1 12

FANCM: 5'-end fancm 10

figl1; fancm 8 (6 calli)

top3a; fancm

Top3a: 3'-end

12 30: 4 ST 5 NS

wt 5

top3a 13

FANCM: 5'-end fancm 4

top3a; fancm 8 (6 calli)

recql4; fancm

RecQl4: 5'-end

50 151: 42 ST 22 NS

wt 77

recql4 32

FANCM: 5'-end fancm 27

recql4; fancm 15 (14 calli)

FIGL1 RecQl4 Top3a

1) Gene knockouts Some lines have no

seeds(NS) or semi-sterile (ST) Cas9 cytotoxicity? GC content: Problem in PCR-

based screening & differential amplification issue

2) Gene replacement attempt 100 embryos bombarded

with SSO then immediately re-transformed with Agrobacterium/CRISPR-FANCMgRNA2.

Plant regeneration without selection.

PCR-based screening.

Saccharification?

CRISPR/Cas9 in 2nd generation biofuel production

CRISPR/Cas9 Knockouts of lignin genes in barley (diploid)

Arabidopsis T-DNA mutants (suppressors)

Barley TILLING mutants Tobacco

lignin genes RNAi

Barley

Lignin biosynthesis pathway.

(1,3;1,4)-b-Glucan

(Kelly Houston,

Guillermo Garcia Gimenez &

Miriam Schreiber)

Other target pathways

Construct Hetrozygous lines %

HvCslF3 6/17 35.3

HvCslF6 4/43 9.3

HvCslF9 13/59 22.0

HvCslH 3/64 4.7

Development

of barley

inflorescence

(Sarah McKim &

Jennifer Shoesmith)

Aphids (Rhopalosiphum padi) resistance in barley (Juronn Bos & Carmen Escudero) LEA gene: 76% mutants Thionins: 78% mutants

R S R L

Conclusions and future work: Gene manipulation in barley (and wheat)

GP <------------------------------- GPMX lines -------------------------------------- > Morex

TILLING in barley optic and Golden Promise

CRISPR/Cas9 works well in barley Golden Promise

Optimisation of gRNA, inducible promoters and Indel screening

Gene replacement currently tested.

Transformation of other cultivars?

Regeneration QTLs (Jennifer Stephens)

Embryogenesis stimulation genes

CRISPR/Cas9 based gene editing in potato (Jennifer Stephens)

gRNA design: high polymorphism

Promoters for Cas9 expression: CaMV 35Sp and Ubiquitinp?

Polyploidy: transgene-free methods?

Transformation efficiency?

callus development shoots

internodes leaf segments microtubers

Maintained in tissue culture

Agrobacterium-mediated Transformation of Potato

Target pathways:

- Biotic/abiotic stresses

- Circadian rhythm

- Alternative splicing

- Small RNAs

- Dormancy

- Food quality

JHI_GT: Dr Pete Hedley Mrs Jenny Morris Ms Clare Booth Mss Louise Donnelly JHI_FUNGEN: Dr Jennifer Stephens Mrs Diane Davidson Mr Jimmy Dessoly JHI_Glasshouses: Mr Alasdair Munro Mr Loeis Speirs Other JHI_Staff: Dr Craig Simpson Dr Kelly Houston Dr Mark Taylor Dr Louise Shepherd Dr Csaba Hornyik

SHUFFLE GROUP

Acknowledgments

Dr Emma Wallington Community Resource for Wheat Transformation, BBSRC Bioinformatics and Biological Resources Fund

Prof Wendy Harwood Dr Mark Smedley

Prof Claire Halpin Mr Sebastian Vivera Dr Miriam Schreiber Dr Christopher McClellan Dr Sarah Mckim Mss Jennifer Shoesmith Dr Juronn Bos Mss Carmen Escudero

Prof Holger Puchta