crispr agbio workshop london...

Vladimir Nekrasov

CRISPR AgBio WorkshopLondon 2017

Nicotianabenthamiana

Tomato Barley

Maize Brassica

Wheat

Model plant: Crop plants:

Applying CRISPR-Cas9 technology in model and crop plants

Arabidopsisthaliana

The pipeline of targeted mutagenesis in plants using theCRISPR/Cas9 system

Belhaj et al. Current Opinion in Biotechnology (2015)

Targeted modification of a plant genome is achieved via harnessingDNA double strand break (DSB) repair pathways:

Adapted from Voytas D. Annu. Rev. Plant Biol. (2013)

1. Non-Homologous End Joining (NHEJ):small random deletions or insertions introduced

2. Homology-Directed Repair (HDR):specified locus modification introduced

The key to targeted genome modification is to have means of introducing a DSB at a specific locus

Single locus mutation

CRISPR/Cas9 applications in plants:targeted genome modification via DNA double strand break (DSB) repair by the non-homologous end joining (NHEJ)

5

Large scale chromosomal deletion

4 5 6

Cas9/sgRNA 1 Cas9/sgRNA 2

1

1 2 3

2 5 6

4 5 6

Cas9/sgRNA 1

1 2 3

5 61 2 3 4

http://www.rgenome.net

CRISPR RGEN Tools is a webtool for designing CRISPR/Cas targets and finding off-targets in genomes

http://www.rgenome.net/

73 plant genomes are in the database

http://www.rgenome.net/cas-offinder/

http://www.rgenome.net/cas-offinder/

PCR

Mutant (biallelic)

Decompositionusing TIDE webtool

Sanger sequencing

Methods for detecting CRISPR/Cas9-induced mutations

https://tide.nki.nl/

https://tide.nki.nl/

PCR with barcoded oligosMutant 1

Next GenerationSequencing (NGS)

Mutant 2

PCR

Mutant (biallelic)


Sanger sequencing


http://crispresso.rocks/

http://crispresso.rocks/

Fluorescent PCRCapillary electrophoresis

high-resolution fragment analysis (HRFA)

PCR with barcoded oligosMutant 1

Next GenerationSequencing (NGS)

Mutant 2

PCR

Mutant (biallelic)


Sanger sequencing


Detection of induced mutations in the GBSS gene by high-resolution fragment analysis (HRFA) using capillary electrophoresis

+

+

PCR, denaturation,annealing

PCR,denaturation

Surveyor assayCelI, T7

High ResolutionMelting analysis

Heteroduplex Mobility Assay (non-denaturing PAGE)

Single-strand conformationpolymorphism (SSCP)(non-denaturing PAGE)


High Resolution Melting analysis

PCR,digesting with a restriction enzyme

Restriction enzymesite loss assay

+

+


PCR,denaturation






Transgenic N. benthamiana

Plant: 2 3

Arrowhead ( ) – MlyI-resistant bandAsterisk (*) – MlyI star activity band

Restriction enzyme site loss assay

Nekrasov et al. Nat. Biotech. (2013)

PCR,digesting with a restriction enzyme

Restriction enzymesite loss assay

+

+


PCR,denaturation





PCRAmplified fragmentlength polymorphism(AFLP)


LBP1 ORF1 T1 P2 ORF2 T2 P3 ORF3 T3 P4 ORF4 T4 P5 ORF5 T5

P6 ORF6 T6 P7 ORF7 T7 P8 ORF8 T8

P11 ORF11 T11

P9 ORF9 T9

P10

1 2 3 4 5

6 7 8

11

9

10 ORF10 T10

Level 2multigeneconstructs

35S ORF Ter

Level 1transcription

units

Level 0Choice ofmodules

P ORF Ter

Promoter 5' UTR ORF Ter

P1 Ter106

P2 Ter107

P3 Ter128

P4 Ter139

P5 Ter1410

Ter6

Ter7

Ter8

Ter

Ter9

Ter1

Ter2

Ter3

Ter4

Ter5

ORF1

ORF2

ORF3

ORF4

ORF5

ORF6

ORF7

ORF8

ORF9

ORF10

ORF11

ORF12

ORF13

ORF14

ORF15

P6

P7

P8

P9

P10

P11

P12

P13

P14

P15

1

2

3

4

5

SP1

SP2

SP3

SP4

SP5

SP6

SP7

SP8

SP9

SP10

SP11

SP12

SP13

SP14

SP15

Libraries of basicmodules

Promoters 5' UTR ORF TerminatorsSignal Peptides

RB

The Golden Gate cloning system allows easy assembly of multiple expression units

In collaboration with S. Marillonnet

GCAATTAC

ACTA

Kan

LB RB

+

Carb

NPTII

GGGA

Red

TGCC

TGCC

Bp

iI

GCAAB

piI

Bp

iI

Bp

iI

BpiI + ligase

+

Carb

Cas9Bp

iI

Bp

iI

ACTA

+

Carb

sgRNA1Bp

iI

Bp

iI

+

pICH47732::NOSp::NPTII-OCSTLevel 1

pICH47742::35Sp::Cas9-NOSTLevel 1

pICH47751AtU6p::sgRNA1Level 1

pAGM4723Level 2 vector

RBGGGA

Kan

LB

Cas9NPTII

ACTA

sgRNA1

TTACGCAATGCC

NPTII-Cas9-sgRNALevel 2

select white colonies

CAGA GGGA

Bp

iI

Bp

iI

Spec

L4E

pICH41780Linker

CAGATTAC

Carb

sgRNA2Bp

iI

Bp

iI

pICH47761AtU6p::sgRNA2Level 1

+

sgRNA2

CAGA

L4E

Using the Golden Gate cloning system to assemble CRISPR constructs

Generating a knockout deletion in the SlMlo1 locus of the Moneymaker tomato using CRISPR/Cas

500 -

400 -

300 -

bp

200 -

1 2 83 4 5 6 7 9 10* * *

500 -

400 -

300 -

bp1 2 8 10WT

* * *

ACATAGTAAAAGGTGTACCTGTGGTGGAGACTGGTGACCATCTTTTCTGGTTTAATCGCCCTGCCCTTGTCCTATTCTTGATTAACTTTGTACTCTTTCAGG

ACATAGTAAAAGGTGTACCTGTGGTGGAGACTGGTGACCATCTTTTCTGGTTTAATCGCCCTGCCCTTGTCCTATTCTTGATTAACTTTGTACTCTTTCAGG

ACATAGTAAAAGGTGTACCTGTGGTGGA------------------------------------------------CTTGATTAACTTTGTACTCTTTCAGG -48



ACATAGTAAAAGGTGTACCTGTGGTGGA-------------------------------------------------TTGATTAACTTTGTACTCTTTCAGG -49

WTPlant 1Plant 2Plant 8

Plant 10

PAM PAMTarget 1 Target 2

***

over 80% of T0 plants carry deletions

Nekrasov et al. Scientific Reports (In Press)

slmlo1

SlMLO1 (WT)

CRISPR/Cas-engineered slmlo1 tomato line is fully resistant to the powderymildew pathogen Oidium neolycopersici

T1 generation slmlo1 plants were screened for the absence of T-DNA

500 -400 -

300 -

8-1

500 -400 -

300 -

bp 8-2 8-3 8-4 8-5 WT

T-DNA

slmlo1

8-6

SlMlo1

T-DNA

slmlo1

WT

slmlo1 8-2

slmlo1 8-4

slmlo1 8-6 (T-DNA)

Illumina reads matching the T-DNA

T-DNA

LB RB


Illumina whole genome sequencing confirmed presence of a homozygous deletion in the SlMlo1 locus

WT

slmlo1 8-2

slmlo1 8-4

slmlo1 8-6 (T-DNA)

WT

slmlo1 8-2

slmlo1 8-4

slmlo1 8-6 (T-DNA)


Target sequence (PAM is in Green) Sites chromosome position off-target sequence Reference

base

Called base

(slmlo1 8-2)

Rate of the

called base

Mutation? Different

from wild

type?

Reference base Called base

(wild type)

Rate of the

called base

Mutation?

CCAATTCTTGATTAACTTTGTAC 1 chr0 15022112 CCAATTCTTtATTAttTTTGTAC C C 0.995 - - C C 0.992 -

2 chr0 15022113 CCAATTCTTtATTAttTTTGTAC C C 0.989 - - C C 0.985 -

3 chr0 15022114 CCAATTCTTtATTAttTTTGTAC A A 1 - - A A 1 -

4 chr0 15022115 CCAATTCTTtATTAttTTTGTAC A A 0.995 - - A A 1 -

5 chr0 15022116 CCAATTCTTtATTAttTTTGTAC T T 1 - - T T 1 -

6 chr0 15022117 CCAATTCTTtATTAttTTTGTAC T T 0.925 - - T T 0.947 -

7 chr0 15022118 CCAATTCTTtATTAttTTTGTAC C C 1 - - C C 0.985 -

8 chr0 15022119 CCAATTCTTtATTAttTTTGTAC T T 0.995 - - T T 0.993 -


10 chr0 15022121 CCAATTCTTtATTAttTTTGTAC t T 1 - - t T 0.993 -

11 chr0 15022122 CCAATTCTTtATTAttTTTGTAC A A 0.995 - - A A 0.993 -


13 chr0 15022124 CCAATTCTTtATTAttTTTGTAC T T 0.995 - - T T 1 -

14 chr0 15022125 CCAATTCTTtATTAttTTTGTAC A A 0.995 - - A A 1 -

15 chr0 15022126 CCAATTCTTtATTAttTTTGTAC t T 0.995 - - t T 0.986 -

16 chr0 15022127 CCAATTCTTtATTAttTTTGTAC t T 1 - - t T 0.98 -



19 chr0 15022130 CCAATTCTTtATTAttTTTGTAC T T 0.995 - - T T 1 -

20 chr0 15022131 CCAATTCTTtATTAttTTTGTAC G G 0.995 - - G G 0.988 -


22 chr0 15022133 CCAATTCTTtATTAttTTTGTAC A A 0.99 - - A A 0.987 -

23 chr0 15022134 CCAATTCTTtATTAttTTTGTAC C C 1 - - C C 0.994 -

Off-target analysis: 145 putative off-targets with up to 4 mismatches analysed

Result: no off-target mutations detected suggesting that CRISPR/Cas is a highly precise tool in tomato


Transform with Cas9/sgRNAs

Callustissue

T0 plantlets

T1 seeds slmlo1 T-DNA segregating

Screen forhomozygous slmlo1 T0 mutants

Screen T1 generationfor T-DNA-free plants

T2 seedsslmlo1T-DNA-free

0 3 6 9.5Time, months3 months 3 months 3.5 months

Homozygous T-DNA free slmlo1 tomato lines have been producedin less than 1 year

Targeted modification of a plant genome is achieved via harnessingDNA double strand break (DSB) repair pathways:

Adapted from Voytas D. Annu. Rev. Plant Biol. (2013)

1. Non-Homologous End Joining (NHEJ):small random deletions or insertions introduced

2. Homology-Directed Repair (HDR):specified locus modification introduced

CRISPR/Cas9 technology applications involving homologous recombination (HR)

• Targeted gene insertion (e.g. GFP tag)• Targeted gene replacement (introducing SNPs into a desired gene)

Targeted Gene Insertion

Gene 1

Gene 1

Repair template

Target locus

Targeted Gene Replacement

Cas9/sgRNA

Gene 1

Gene 1

Target locus

Repair templateCas9/sgRNA

GFP

GFP

Cas9/sgRNACas9/

sgRNACas9/sgRNA

Adapted from Schiml et al. Plant J. (2014)

Targeted insertion of the nptII selectable marker into ArabidopsisADH1 locus using homologous recombination (HR)

LB

Cas9 sgRNA

ADH1

Cas9

LB RBnptIIsgRNA

nptII

RBADH1

Cas9LB RB

nptIIsgRNA

Selected on kanamycin and allyl alcohol

ZmALS2

ZmALS2

Cas9/sgRNA

Adapted from Svitashev et al. Plant Physiology (2015)

Cas9

ZmALS2

ZmALS2P165S+

DNA repair template

Plant cell nucleus

sgRNA

ZmALS2P165S

ZmALS2P165S

ZmALS2P165S

Maize resistant to chlorsulfuron

WTALS2P165S

Chlo

rsulfuro

n(1

00

µg

L−1)

HDR-mediated ZmALS2 allele replacement in maize using CRISPR/Cas9

GOI1P T

Chromosome

GOI1P T GOI2P T

GOI1P T GOI2P T GOI3P T

LP

LP

LP

LP

Generating a “Landing Pad” (LP) for inserting genes of interest (GOIs) at aspecified location on a chromosome

Advantage: one can avoid the position effect when expressing transgenes

BARP T exon2 HPT Tintron Chromosome

Cas9P T

P intronexon1 HPT

sgRNAP

GOIP T

DNA repair template/ Cas9 + sgRNA vector

P exon1 HPTGOIP T exon2 HPT Tintron Chromosome

BAR – Basta selectable marker; HPT – Hygromycin selectable marker; GOI – gene of interest; P – promoter; T – terminator

Generating a “Landing Pad” for inserting genes of interest at a specifiedlocation on a chromosome

CRISPR/Cas system

Genome Editing As A Tool To Speed Up Breeding Of Crops With Improved Agronomic Traits

Advantages of genome editing as compared to traditional breeding:

• Speed: mutations can be introduced directly into an elite variety without lengthy crosses with e.g. a related wild species and then backcrosses to the elite line

• No linkage drag: unlike in conventional breeding, a mutation can be introducedinto an elite variety without closely linked genes that may confer an adversephenotype

• Precision: CRISPR practically does not generate off-target mutations, while e.g. EMS mutant lines have thousands of background mutations

• Multiplexing: ability to target a few genes at the same time (e.g. homologues or genesarranged in a gene cluster)

Challenges for CRISPR/Cas-assisted crop breeding:

• need to broadly connect genotype with phenotype - Gene function predictions based on model species e.g. Arabidopsis

- GWAS on accessions that show variation in the trait of interest- Forward genetic screen (e.g. using EMS-mutagenized population)

• aim to develop the technology to level when multiple precise genetic changes can be simultaneously introduced into an elite crop variety

Genetic locus fine-mapping

The Sainsbury Laboratory (Norwich)

Sophien Kamoun

Joe Win

and the rest of the Kamoun Lab

Jonathan Jones

Earlham Institute

Nicola Patron

Oleg Raitskin

Cold Spring Harbor Laboratories

Zachary Lippman

Chris Brooks

Boyce Thompson Institute

Joyce van Eck

MPI Tubingen

Detlef Weigel

Institute of Digital Agriculture,

Zhejiang Academy of Agriculture

Sciences

Congmao Wang

Acknowledgements

Achim DobermannMalcolm Hawkesford

Caroline SparksDamiano MartignagoLucy Hyde

crispr agbio workshop london...

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