A CRISPR/Cas9-mediated gene editing to enhance the ... Scarano DiSBA 2017.pdf · ISPA-CNR, Lecce,...
Transcript of A CRISPR/Cas9-mediated gene editing to enhance the ... Scarano DiSBA 2017.pdf · ISPA-CNR, Lecce,...
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 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