Large scale genome editing for. Senior Scientist, GenScript

Transcription

1 Large scale genome editing for metabolic engineering of E. coli YifanLi Li, Ph.D PhD Senior Scientist, GenScript

2 Metabolic engineering Cell factory Remove inhibition Substrate Overexpressing pathway genes A B C D E F Blocking competing pathways M Introducing exogenous genes Make Research Easy 2

3 λ red recombination system λ Red recombineering is a common technique for genome editing in bacterial cells Exo has a 5 - to 3 -dsdna exonuclease activity Beta binds the single-stranded DNA Gam (not shown here), which prevents RecBCD nucleasefrom degrading double-stranded linear DNA fragments Make Research Easy 3

4 Traditional λ Red recombineering Up λ Red recombination Marker Down Replaces a specific chromosomal sequence with a selectable antibiotic resistance gene KO Gene selectable seectabe marker is flanked by two frt site Markers are removed by FLP-FRT frt frt Disadvantages: 2 recombination steps and selectable marker required Scar can impact subsequent generations Make Research Easy 4

5 Selection/counter-selection strategy for seamless editing Up selection/counte r-selection cat sacb Down A counter-selectable cassette is inserted into the targeting g site along with the selectable marker KO Gene A counter-selectable maker helps to select the recombinant strains that have lost the marker in the second recombination Disadvantages: 2 recombination steps and selectable marker required Time and labor consuming Make Research Easy 5

6 λ Red + I-SceI strategy for seamless editing Up λ Red + I-SceI The λ Red recombineering technology has been combined with Marker Down I-SceI cleavage SceI KO Gene SceI I-SceI recognition sites are flanked upstream and downstream of marker, and the I-SceI endonuclease makes DSBs at these sites Result: KO without a scar Disadvantages: 2 recombination steps required Still need a selectable marker Make Research Easy 6

7 CRISPR mediated genome editing The functionality of CRISPR relies on Cas9 and grna CRISPR system introduces double strand breaks at targeted loci DSB can be repaired through Non- Homologous End Joining (NHEJ) or Homology Directed Repair (HDR) Combining of CRISPR and Red system allows efficiency genome editing in E. coli Make Research Easy 7

8 GenScript s λ Red CRISPR system λ Red - CRISPR/Cas Up Down Simple and efficient :Red-CRISPR only requires one recombination event and no selectable marker is required KO Gene CRISPR generated Double Strand Break Recombinant selection is easy: double strand breaks generated by CRISPR are lethal without a recombination event Red-CRISPR technology enables multigene editing for expanded applications Make Research Easy 8

9 Genome editing workflow Gene synthesis (KI/replacement) DNA synthesis and cloning grna design and construction grna design and construction using algorithm developed at Broad Institute Donor DNA construction CRISPR genome editing λ Red- CRISPR/Cas9 editing QC PCR screening and sequencing of recombinant strains Make Research Easy 9

10 Service Details Gene knock-out, knock-in or gene replacement in E. coli Catalog # Service Deliverables Timeline Starting Price SC1730 Knock-out strain generation SC1731 Customer provides: Starting strain Target gene name Target gene sequences, if the whole genome sequence is unavailable Knock-in strain generation Recombinant bacterial strains in the format of glycerol stock Starting from 4 weeks $4,000 Customer provides: Starting strain Target gene sequence* Sequence q of the target engineered region, if the whole genome sequence is unavailable QC report Starting from 4 weeks $5,400** *standard gene synthesis (SC1010) can be used for the KI gene insert **pricing does not include additional cost for gene synthesis, if required Make Research Easy 10

11 Metabolic engineering for β- carotene production MEP pathway G3P dxs dxr ispd ispe ispf ispg DXP MEP CDP-ME CDP-MEP MEC HMBPP isph IPP idi ispa FPP Pyr DMAPP β-carotene synthetic pathway crte β-carotene crty Lycopene crti Phytoene crtb GGPP Make Research Easy 11

12 Integrating crtebiy genes into the genome of E. coli crte crtb crti crty crtb crti crte crty ldha CRISPR cutting Chromosome integration Stable No antibiotic selection Plasmid expression Unstable Antibiotic selection required Make Research Easy 12

13 Overexpressing MEP pathway genes G3P Strong Promoter Strong RBS Nine-gene MEP pathway FPP Pyr Native RBS ORF Make Research Easy 13

14 What if we don t know the best expression level Production Production Production Expression Expression Expression Product Product Product Growth Growth Growth Make Research Easy 14

15 Using RBS library to tune gene expression level RBS 1 RBS 2 RBS 4 RBS 5 RBS 3 RBS 6 RBS Library Strength [au] Native RBS ORF RBS 1 1 RBS 2 10 RBS RBS RBS Make Research Easy 15

16 Red-CRISPR for metabolic engineering g Gene Knock out Gene Overexpression Gene Gene Knock in Tuning Gene Expression Gene Make Research Easy 16

17 Metabolic Pathway Assembly Service Based on OLMA technology Research Strategy Recommended Service Deliverable Format How to Order Rational Design Library Screening Metabolic Pathway Assembly Constructs Cat. No. SC1702 Metabolic Pathway Assembly Library Cat. No. SC1707 Individually sequence-verified plasmids, 4µg per construct Price and turnaround depend upon sequence length, complexity, and number of constructs desired. 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 diversity it of pooled library. Request a Quote for your customized project Make Research Easy 17

18 Q&A If you have any other questions, visit Or or Make Research Easy 18