Dr. Aurelia Scarano

Napoli, 22 Dicembre 2017

A CRISPR/Cas9-mediated gene editing to enhance the expression

of the Solanum lycopersicumMYB12 gene and the nutritional

value of tomato

Phenylalanine

4-Coumarate-Coa

Naringenin Chalcone

Naringenin

Dihydroflavonols

Flavonols

Anthocyanins

Stilbenes

Aurones

Flavones

Phenolic acids

Flavanols

BENEFITS FOR THE HEALTH

• Antioxidant activities

• Anti-inflammatory properties

• Healthy aging

• Protection against

inflammatory and chronic

diseases

Isoflavones

Proanthocyanidins

In tomato, polyphenols are present only in the skin (1% of the whole fruit)

• Improvement of polyphenol content with conventional

breedings

• Metabolic engineering is a useful tool to increase the levels

of polyphenols in crops, such as tomato

AmDelila

(bHLH)

AmRosea1

(MYB)

Phenylalanine

4-Coumarate-Coa

Naringenin Chalcone

F3H

Naringenin

Dihydrokaempferol

CHI

CHS

PAL

Phenolic acids

F3’5’HF3’H

FLS

Kaempferol

Dihydromyricetin

Myricetin

Dihydroquercetin

FLS

Quercetin

Rutin

GT

DFR

ANS

3GT

RT

5GT

DFR

ANS

3GT

RT

5GTPelargonidinsCyanidins Petunidins

AtMYB12

DFR

ANS

3GT

RT

5GT

Over-expression

under the fruit-

specific E8

promoter

Phenylalanine

4-Coumaroyl-CoA

Naringenin Chalcone

F3H

Naringenin

Dihydroflavonols

CHI

CHS

PAL

Flavonols Anthocyanins

Resveratrol Piceid

VvStSy

Stilbenes

WT

35S::StSy

ISPA-CNR, Lecce, Italy (Dr. Angelo Santino) and John Innes Centre, Norwich, UK (Prof. Cathie Martin)

Anthocyanins

Flavonols

Flavonols Stilbenes

Flavonols Anthocyanins Stilbenes

AmDel/Ros1

AtMYB12

AtMYB12/AmDel/Ros1

AtMYB12/AmDel/Ros1/VvStSy

WT

AtMYB12/VvStSy

Flavonols Anthocyanins

Diets enriched in flavonols, anthocyanins and stilbenes

alleviate the symptoms of gut inflammation

Start DSS treatment

DSS- inducedinflammation as a model

of IBD (InflammatoryBowel Disease)

Antioxidant capacity in Bronze 7-fold higher than in WT

Scarano A., Santino A., et al. 2017, Frontiers in Nutr, in revision

genome editing technologies

The CRISPR/Cas9 systemSite-specific mutations on the target gene, using Cas9 endonuclease

Cas9 is driven by RNA-guide/s to

recognize a target PAM sequence

and cut 3 nucleotides upstream

NHEJ (Non-homologous

end joining)

HR(Homologous

recombination)

Small deletions or small insertions

donor template

Knocked-in sequence of interest

+

chromatids

insertion of the 35S promoter ANT1 gene

Viral sequences

Cermak T. Genome Biology 2015

Insertion E8 promoter upstream MYB12 gene

homologous recombination (via HR)

Stable transformation with Agrobacterium tumefaciens

Transient expression assays

E8 promoterHomology arm Homology armNPTII Cas9 LIR RepLIR

LB RB

sgRNA

SlMYB12 promoter SlMYB12 gene

CTATTTATTGGCATTTCTATTGG

Gene of interest: SlMYB12 (involved in the flavonoid pathway)

RNA guide

G6F6

LEFT JUNCTION

WT F6 B8 H6

RIGHT JUNCTION

1/57: left and right junction

1/57: left junction

2/57: right junction

• Transformation with Agrobacterium rhizogenes: 57 transformed/ 60 hairy roots

• Screening to detect gene targeting events

Flanking genomic

region

Flanking genomic

region

EBA F

MYB12 promoter(Homology)

MYB12 gene(Homology)

E8 promoter

LEFT JUNCTION RIGHT JUNCTION

CRISPR/Cas9 editing - via HR:

WT G6 F6 B8 H6

off-target #2

WT

G6

F6

B8

H6

WT G6 F6 B8 H6

off-target #1

WT

G6

F6

B8

H6

Off target 1 (strand +): ACTTTTATTGGCATTTCAATTGGOff target 2 (strand -): ATGTTTATTAGCGTTTCTATTGG

CTATTTATTGGCATTTCTATTGG

off-target #3

WT G6 F6 B8 H6 WT G6 F6 B8 H6

off-target #4

Off target 3 (strand -): TTTTTTATTGGAGTTTCTATGGGOff target 4 (strand +): CTATCTACTGTCATTTCTAAAGGOff target 5 (strand +): GTATTTATTGAGATTTCAATAGG

CTATTTATTGGCATTTCTATTGG

WT

G6

F6

B8

H6

WT

G6

F6

B8

H6

WT G6 F6 B8 H6

off-target #5

WT

G6

F6

B8

H6

NPTII 35S:Cas9

LB RB

sgRNA E8 promoter

Site guide Site guide

NPTII Rp5Sa:Cas9

LB RB

sgRNA E8 promoter

Site guide Site guide

1

2

Hairy roots expression assays

Stable transformation with Agrobacterium tumefaciens

No off-target activities

CRISPR/Cas9 editing - via HR:

CRISPR/Cas9 editing - via NHEJ:

Stable transformation with Agrobacterium tumefaciens

Prof Cathie Martin, Dr Eugenio Butelli

Dr Angelo Santino

Thank you!Dr Fabio D’Orso