crispr/cas9 based gene editing in barley & potato: from...
TRANSCRIPT
CRISPR/Cas9 Based Gene Editing in Barley & Potato: From Construct Design to Mutation
Screening
Abdellah Barakate
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
Exp
erim
enta
l sy
stem
s
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