large scale genome editing for metabolic engineering of e ... · mep pathway dxs dxp mep cdp-me...
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L l diti fLarge scale genome editing for metabolic engineering of E. colimetabolic engineering of E. coli
Yifan Li Ph DYifan Li, Ph.DSenior Scientist, GenScript
Metabolic engineering
Cell factoryRemove inhibition
Substrate Overexpressing pathway genes
A B C D E F
Introducing exogenous genesBlocking
ti Mcompeting pathways
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λ red recombination system
λ Red recombineering is a common technique for genome editing intechnique for genome editing in bacterial cells
Exo has a 5’- to 3’-dsDNA Exo has a 5 to 3 dsDNA exonuclease activity
Beta binds the single-stranded DNAg
Gam (not shown here), which prevents RecBCD nucleasefrom degrading double-stranded linear DNA fragments
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Traditional λ Red recombineering
Replaces a specific chromosomal sequence with a selectable antibioticM kU
λ Red recombination
sequence with a selectable antibiotic resistance gene
selectable marker is flanked by two
MarkerUp Down
se ectab e a e s a ed by t ofrt site
Markers are removed by FLP-FRT
KO Gene
y
Disadvantages:• 2 recombination steps and
frtfrt
pselectable marker required
• Scar can impact subsequent generations
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Selection/counter-selection strategy for seamless editing
A counter-selectable cassette is inserted into the targeting site along
gy gselection/counte
r-selection g g g
with the selectable marker
A counter-selectable maker helps to
sacBUp Downcat
select the recombinant strains that have lost the marker in the second recombination
KO Gene
Disadvantages:• 2 recombination steps and
selectable marker requiredselectable marker required• Time and labor consuming
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λ Red + I-SceI strategy for seamless editing
λ Red + I-SceI The λ Red recombineering technology has been combined with
MarkerUp Down
KO Gene
I-SceI cleavage
I-SceI recognition sites are flankedt d d t f k
SceISceI
upstream and downstream of marker, and the I-SceI endonuclease makes DSBs at these sites
Result: KO without a scar
Disadvantages: Disadvantages:• 2 recombination steps required• Still need a selectable marker
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CRISPR mediated genome editingg
The functionality of CRISPR relies The functionality of CRISPR relies on Cas9 and gRNA
CRISPR system introduces double CRISPR system introduces double strand breaks at targeted loci
DSB can be repaired through Non-p gHomologous End Joining (NHEJ) or Homology Directed Repair (HDR)
Combining of CRISPR and Red system allows efficiency genome editing in E. coli
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GenScript’s λ Red – CRISPR system
Simple and efficient :Red-CRISPR only requires one recombination
λ Red -CRISPR/Cas y q
event and no selectable marker is required
Up Down
Recombinant selection is easy: double strand breaks generated by CRISPR are lethal without a
KO Gene
recombination event
Red-CRISPR technology enables
CRISPR generated Double Strand Break
Red CRISPR technology enables multigene editing for expanded applications
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Genome editing workflow
Gene synthesis (KI/replacement)
• DNA synthesis and
gRNA design and construction
• gRNA design and
CRISPR genome editing
•λ Red- CRISPR/Cas9 editing
QC
• PCR screening and sequencing of
cloning construction using algorithm developed at Broad Institute
• Donor DNA construction
editingrecombinant strains
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Service Details
Catalog # Service Deliverables Timeline Starting Price
Gene knock-out, knock-in or gene replacement in E. coli
SC1730 Knock-out strain generation
Customer provides:•Starting strain
Starting from 4 weeks
$4,000•Target gene name•Target gene sequences, if the whole genome sequence is unavailable
Recombinant bacterial strains in the format of
glycerol stock
4 weeks
SC1731 Knock-in strain generation
QC reportCustomer provides:•Starting strain•Target gene sequence*•Sequence of the target engineered
Starting from 4 weeks $5,400**
q g gregion, if the whole genome sequence is unavailable
*standard gene synthesis (SC1010) can be used for the KI gene insert
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**pricing does not include additional cost for gene synthesis, if required
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Metabolic engineering for β-carotene production
MEP pathway
dxsDXP MEP CDP-ME CDP-MEP MEC HMBPP
dxr ispD ispE ispF ispGFPP
ispAG3P IPP
ispHidi
Pyr DMAPP
β-carotene synthetic pathway crtE
GGPPPhytoeneLycopeneβ-carotenecrtBcrtIcrtY
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Integrating crtEBIY genes into the genome of E. coli
crtE crtB crtI crtY tB tI
crtE
crtE crtB crtI crtY crtB crtI
crtY
ldhA
CRISPR tti
Chromosome integration Plasmid expression
CRISPR cutting
•Stable•No antibiotic selection
•Unstable•Antibiotic selection required
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Overexpressing MEP pathway genes
StrongPromoter
StrongRBS
FPP
G3P
Nine-gene MEP pathway
NativeRBS
ORFPyr
p y
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What if we don’t know the best expression level
Production Production ProductionProduction Production Production
E i E i E iExpression Expression Expression
Product Product Product
Growth
Product
Growth
Product
Growth
Product
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Growth Growth Growth
Using RBS library to tune gene expression level
RBS 1
RBS 2
RBS 4
RBS 5
StrengthRBS
RBS 3 RBS 6
RBS 1
[au]
1
Library ORFNative RBS
RBS 2
RBS 3
10
100
RBS 4
RBS 5
1000
10000
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Red-CRISPR for metabolic engineeringg g
Gene Knock out Gene Overexpression
G K k i G
Gene
Gene Knock in Tuning Gene Expression
Gene
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Gene
Metabolic Pathway Assembly ServiceBased on OLMA technology
Research Strategy
Recommended Service Deliverable Format How to Order
Rational Design
Metabolic Pathway Assembly –ConstructsCat. No. SC1702
•Individually sequence-verified plasmids, 4µg per construct•Price and turnaround depend upon sequence length, complexity, and number of constructs desired.
R t Q t fRequest a Quote for your customized projectLibrary
ScreeningMetabolic Pathway Assembly –LibraryCat. No. SC1707
•10 μg of pooled plasmids (ligation products)•Price and turnaround time depend upon sequence length, complexity, theoretical library size, and validation requests e.g. restriction analysis and sequencing to confirm accuracy and di it f l d lib
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diversity of pooled library.
Q&A
If you have any other questions, visit http://www.genscript.com/CRISPR-microbial-genome-editing.html
http://www.genscript.com/metabolic-pathway-assembly.html
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Or email: [email protected] or [email protected]