CRISPR Is On The Move: Genome Editing From Rice To Wheat

Plant Genomics and Gene Editing Congress16th March 2017

Amsterdam- The Nederlands

CRISPR is on the move: genome editing from rice to wheat

Damiano Martignago

@DamaTheBeast

https://twitter.com/DamaTheBeast

Production of rice florigens KO-mutants

CRISPR is on the move: genome editing from rice to wheat Damiano Martignago #PGGEC17 16th March 2017

HD3A exon1 GGACGCGTTCG-TCCGGAGCACCAACC-TCAAGGTCA

HD3B exon1 GGATCCATTCG-TCCGGATCACTAACC-TCAGTGTCA

*** *.****-****** *** ****-***. ****

HD3A

Plant1.1 GGACGCGTTCG-TCCGGAGCACCAACCCTCAAGGTCA +1

Plant1.2 GGACGCGTTCG-TCCGGAGCACCAACCCTCAAGGTCA +1

Plant2.1 GGACGCGTTCG-TCCGGAGCACCAAC--TCAAGGTCA -1

Plant2.2 GGACGCGTTCG-TCCGGAGCAC----C-TCAAGGTCA -4

Plant3.1 GGACGCGTTCG-TCCGGAGCACCAAC--TCAAGGTCA -1

Plant3.2 GGACGCGTTCG---------------------GGTCA -19

HD3B

Plant1.1 GGATCCATTCGATCCGGATCACTAACC-TCAGTGTCA +1

Plant1.2 GGATCCATTCGTTCCGGATCACTAACC-TCAGTGTCA +1

Plant2.1 GGATCCATTCGATCCGGATCACTAACC-TCAGTGTCA +1

Plant2.2 GGATCCATTCG-------TCACTAACC-TCAGTGTCA -6

Milan

Harpenden

HD3A

HD3B

Amsterdam

Courtesy of Jorge Gomez-Ariza

Wheat is more challenging


Transformation protocol • Funnel effect:

• Transformation efficiency 5-20%• CRISPR/Cas9 efficiency 5-7%• Overall efficiency 0.25-1.4%

Wheat-specific hurdles?• Large hexaploid genome• Long generation time• Requirement for wheat-optimised Cas9

Rothamsted Research wheat-specific vectors


pRRes209RRM_482 gRNA expression

cassette

pRRes226RRM_497 RResCas9-RFP

Vector design by A. Huttly

RResCas9 is expressed and localized in wheat cell nucleus


pRResCas9-RFP+

+pRResCas9-RFP

gRNA cassette

H2B-GFP

gRNAs to target a conserved restriction site


• 2 gRNAs • Targeting conserved regions in the ABD genomes• Designed to disrupt conserved SalI restriction sites• ≈100bp interval (potentially causing large deletion)

gRNA1 gRNA2

5’>3’ PAMNGG

3’>5’ PAMCCN

A genome

B genome

D genome

Initial screening of transformed plants


M 1 9 2 10 3 11 4 12 5 13 6 14 7 15 8 16 17 21 18 22 19 23 20 24 M

M 25 41 26 42 27 43 28 44 29 45 30 46 31 47 32 48 33 37 34 38 35 39 36 40 M

49 57 50 58 51 59 52 60 53 61 54 C+ 55 56 C- M

• DNA extraction from regenerated plantlets (leaf)

• PCR with generic (ABD) primers• Non edited amplicon ≈ 650bp • Large deletion ≈ 550bp

• 60 samples checked, no large deletion detected

Mutant screening – RFLP


M Ctrl Und Ed 1 Ed 2 HetCtrl

29 35

10 16 19 20

• Cas9-RFP, plants regenerated with no selection

• Generic primer PCR followed by SalI RFLP

• Screened plants: 60• Putative edited plants identified: 6 + 2

Genome-specific RFLP genotyping


500bp-

M 10 16 20 29 61 35 C+ Ctrl Und M

Genome A Genome B500bp-

100bp-

Genome D

1000bp-

M 10 16 20 29 35 61 C+ Ctrl Und M M 10 16 20 29 35 61 C+ Ctrl Und M

RefSeq

Sample 20

CtrlGenome D CCGCGTCGACTTC

Allele1 CCGCGTCGACTTC


Genome B CCGCGTCGACTTC

Allele1 CCGCGT-GACTTC


Genome A CCGCGTCGACTTC

Allele1 CCGCGT-GACTTC

Allele2 CCGCGT--ACTTC

Summary


M 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 M

M 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 M

M 87 88 89 90 91 92 93 94 95 96 97 98 99 100 C+ C- M

Experiment ID Plant # Mutants Efficiency

3607 100 8 8%

3609 61 6 9.8%

• Wheat optimised Cas9 + tRNA/gRNA system =

• Highly efficient

• No selection protocol: Potentially GM-free Quick plant regeneration Genotyping

Mutant genotyping


• Initial screening: High Resolution Melting

• Mutant characterization with cloning: Precise Time and labour consuming Quite expensive

• Mutant characterization with NGS: Precise Quick Very expensive

Aknowledgments

Alison HuttlyMegan RafterAndy PhillipsSteve ThomasNigel HalfordSarah RaffanCaroline SparksLucy HydeAngela DohertyMandy RileyMelloney St-LegerVladimir NekrasovMalcolm Hawkesford

Keith Edwards

Fabio FornaraJorge Gómez-ArizaVittoria Brambilla

Martina CeriseMicol Aldrovandi

[email protected]

Science

CRISPR Is On The Move: Genome Editing From Rice To Wheat