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: Yifan.Li@genscript.com or Laura.Geuss@genscript.com