Advances within Gene Synthesis and Genome Editing Anja Martinez, PhD Technical Sales Specialist – Synthetic Biology Biosciences, Life Sciences Solutions, Geneart AG, Regensburg, Germany The world leader in serving science 1 Agenda • Gene synthesis • Optimization • Strings DNA Fragments • Directed evolution • Elements • Vector construction • Combinatorial parts assembly • Genome editing • Introduction • Tools 2 Gene synthesis Gene synthesis Optimization Strings DNA Fragments The world leader in serving science 3 GeneArt – Synthetic genes 4 • Synthetic Genes are double stranded DNA constructs, synthesized to the customer specification based on the customers digital sequence • Synthetic Genes can routinely be made >10kb in length • Genes are delivered in a GeneArt® standard cloning vector (pMX series) or the vector of the customer’s choice (additional service) • Standard deliverable is 5 µg lyophilized DNA, larger amounts are available based on additional plasmid preparation (additional service) • All genes are 100% sequence verified prior to shipment and come along with a quality assurance documentation The world leader in serving science 5 Price reductions have increased demand 7000 7,000 GENEART Gene Synthesis capacity / month (kbp) 6000 5,500 >1,500 orders per month All products made-toorder 5000 4000 Ecoli K12 4,369 4,750 4,000 3000 2,500 2000 1,600 1000 10 20 40 80 150 2000 2001 2002 2003 2004 300 ‘Synthia‘ 800 592 500 0 2005 2006 2007 2008 2009 2010 2011 2012 2013 Current capacity: 7 Mbp/month 6 GeneArt GeneOptimizer The world leader in serving science 7 In silico optimization of genes - GeneOptimizer™ Clients‘ specific Requirements & target vector determinations CDS Genetic stability Transcription Process Stability of mRNA Translation Process Optimization of the coding sequence is a multiparameter challenge 8 Multigene study in mammalian HEK293T cells Multiparameter RNA and Codon Optimization: A Standardized Tool to Assess and Enhance Autologous Mammalian Gene Expression 50 standard human genes representing the most interesting protein classes were selected from the NCBI data bank Protein Kinases Transcription Factors Ribosomal Proteins Cytokines Membrane Proteins 9 GeneOptimizer® Expression study of 50 mammalian genes: wildtype vs. optimized (B) PP 3 wt = opt ( 3 ) Jnk3 Kinases (C) 40 relative expression PP 2 PP 1 optimized PP 3 PP 2 Mock PP 1 wildtype (A) Jnk3 ▲ Transcription Factors 40 CREB1 AQP5 30 x 13 15 ▲ x9 IL-2 ▲ absolut ( 7 ) opt > wt SMARCD1 wildtype wildtype = optimized ( 5 ) optimized relative expression SMARCD1 40 ( 16 ) opt > wt optimized wt = opt ( 1 ) wildtype Ribosomal Proteins wt = opt ( 1 ) AQP5 relative expression IL-2 Cytokines ( 4 ) opt > wt optimized wt > opt ( 1 ) wildtype 20 ( 12 ) opt > wt ▲ relative expression 60 x 2.2 CREB1 wildtype Membrane Proteins optimized relative expression wildtype optimized ▲ x 1.3 ( 4 ) opt > wt wildtype > optimized ( 1 ) Yields: 88% of proteins express higher with optimized genes Reliability: 100% of optimized genes were expressed while 12% of wildtype genes showed no detectable expression optimized > wildtype ( 44) Fath et al. (2011) Multiparameter RNA and Codon Optimization: A Standardized Tool to Assess and Enhance Autologous Mammalian Gene Expression. 10 PLoS ONE 6(3): e17doi:10.1371/journal.pone.0017596 GeneArt® Gene Synthesis Benefits wild type sequence GeneOptimizer® pat. pend. Efficiency – De novo synthesis is cost effective and fast Availability – all sequences are accessible, easy to order Flexibility – no restrictions in design, no natural template is required Performance – optimization significantly enhances the expression probability Reliability – GeneArt® technology provides reliable delivery and success rates Service Offering – comprehensive portfolio from GeneArt® Strings™ to proteins www.lifetechnologies.com/genesynthesis 11 Online ordering portal 12 Traditional cloning vs. gene synthesis A typical 330 amino acid protein / 1000 bp gene Classical cloning GeneArt® Gene synthesis service Design & order oligos PCR reaction and purification Cloning kit PCR screening Midi prep Agarose gel analysis Sequencing Order online (design & optimize) Production Shipment (100% sequence verified) Time (33 € per hour) 4 hrs = 132 € + 133 € + 26.6 € Time (33€ per hour) Total cost = 291.6 € in 8-10 days Total cost Expression rate 1 x wild type Expression rate 3-100x wild type Material Failure rate (20%) x1.20 Material Failure rate (0%) 9 min =4,95 € +250 € +0 € =254.95 € in 9 days 13 GeneArt® Strings™ DNA Fragments Your source for genes. Fast and affordable for every lab. The world leader in serving science 14 GeneArt Gene Synthesis Service & Strings DNA fragments Oligo synthesis Oligo assembly PCR amplification Cloning and screening Quality control (batch) Quality control (single clone) Cloning and screening in your hands 15 Properties: GeneArt® Strings™ DNA Fragments What are GeneArt® Strings™ DNA fragments? • Custom-made linear dsDNA fragments, up to 3 kb • Produced with the same technology we use for gene synthesis • You specify 5‘ and 3‘ ends supporting cloning or assembly into larger genes in your lab Price •Up to 62% cheaper than gene synthesis Processing time • 5 to 8 business days, depending on length category Deliverable • >200 ng lyophilized DNA, column purified PCR fragment pool, ready to clone Quality control • Gel electrophoresis • Bulk sequencing to ensure your gene is highly represented Ordering • Online ordering portal with sequence editing and optimization functionality • Multi-sequence batch upload (Large Order Assistant) • Assistant to assemble genes >3 kb from Strings fragments • Excel® file-based ordering possible (no gene editing and optimization support) 16 GeneArt® Strings™ fragments vs Gene Synthesis GeneArt® Strings™ DNA fragments GeneArt® Gene Synthesis Product description Linear dsDNA fragments up to 3000bp, assembled from oligonucleotides and PCR amplified, ready to clone by customer Cloned genes or gene fragments of any length, 100% sequence verified Production time 5-8 business days, Dependent on length category 9 business days (up to 1.2 kb) Deliverable > 200ng dried (linear PCR fragment, ready to use) 5µg dried (gene cloned in plasmid) Ordering Same as for gene synthesis Online order portal with sequence editing and optimization functionality, multi sequence batch upload, direct ordering with immediate price info; Excel based ordering Support Support provided in portal ordering, no support for sequence optimization or editing provided by service team for non portal orders Full support for portal and non portal orders 17 Directed Evolution Directed evolution is a method used in protein engineering that mimics the process of natural selection to evolve proteins or nucleic acids toward a user-defined goal. Wikipedia The world leader in serving science 18 What can Directed Evolution achieve? Raise the affinity of antibodies Enhance the thermostability of industrial enzymes Improved variant Increase the specific activity of an enzyme Wild type Modify selected protein properties at your convenience! 19 How Directed Evolution Works? Mutation Bottle necks (library generation): Example: Protein length 55 aa Number of possible mutants (diversity): 2055 = 3,6*1071 = Number of all atoms in the universe Selection (Screening) Bottle necks (Screening): • RNA-display ~1012 • Phage display ~ 109-1010 • Enzyme assays ~ a few 1000 -> Limit the diversity to a managable but still meaningful size 20 Rational Directed Evolution Workflow Aim: rationally limit the size of the library (diversity) GeneArt® Site-saturation mutagenesis • Systematically screen all single substitution variants of a target protein to identify beneficial substitutions Diversity: 300 aa protein = 300 * 19 = 5700 Screen GeneArt® Combinatorial libraries • Combination of all beneficial substitutions to screen for synergies and an even better protein e.g. 5 site with 3 substitutions each = 35 = 243 Screen Improved variant Outcome • Higher specific enzyme activity • Improved enzyme thermostability Wild type • etc 21 Rational Directed Evolution Workflow Short cuts: • Structural data • Homology data • Literature • etc Screen Improved variant Wild type 22 GeneArt® Directed Evolution Platform for Protein Improvement Site-Saturation Mutagenesis Site-Directed Mutagenesis Introduce single or multiple mutations. Targeted PCR-based process and subcloning into your vector of choice. Systematic replacement of wildtype amino acid by all 19 non-wildtype amino acids. Checks every possible variant at each position. Combinatorial Libraries GeneArt® Directed Evolution Toolbox Truncation Libraries De novo synthesis based on true rational design. High degree of mutagenesis at targeted locations. Controlled Randomization Libraries Incrementally trims genes around defined core. Preserves ORF, avoids out-of-frame mutations. Randomize entire ORF or confined region. Quality Control 23 Applications • Fully synthetic antibody libraries • Affinity maturation of antibodies • Humanization of antibodies/scaffolds • Improvement of scaffolds • Improvement of industrial enzymes • Homology reduction (avoid IP issues) • Change of substrate specificity or enantioselectivity • Alanine Scans -> Functional studies • Generation of alternative splice forms • Fusion of gene to different Tags or leaders • Construction of knock-out constructs • etc. 24 GeneArt® ElementsTM Vector Construction and Combinatorial Parts Assembly The world leader in serving science 25 GeneArt® Elements™ VC: Example from DNA Parts Repository part number public name GA-pro-00016.1 GA-pro-00015.1 GA-cut-00012.1 GA-rep-00001.1 GA-rep-00002.1 GA-cut-00094.1 GA-clv-00002.1 GA-tag-00002.1 GA-cod-00063.1 GA-pla-00001.1 GA-ori-00002.1 GA-pro-00020.1 GA-cod-00001.1 GA-tag-00006.1 GA-tag-00001.1 CMV promoter GA-rca-00003.1 GA-res-00004.1 GA-tag-00007.1 GA-tag-00003.1 GA-cod-00064.1 GA-pla-00002.1 GA-ori-00001.1 GA-rca-00001.1 GA-stu-00001.1 EF-1alpha promoter AscI emerald GFP YFP TOPAZ KpnI TEV recognition site 6x His tag Stop codon TAA BGH polyA site f1 origin SV40 promoter Start codon V5 tag c-myc tag Neomycin resistance cassette for mammalian cells Neomycin resistance orf; npt2 HA tag DYKDDDDK tag Stop codon TGA SV40 polyA site colE1_ori Ampicillin resistance cassette; bla part family/class part category regulation regulation construction CDS CDS construction CDS CDS CDS regulation regulation regulation CDS CDS CDS promoter promoter restriction site reporter reporter restriction site Protease cleavage site tag (purification, detection) codon polyA site replication origin promoter codon tag (purification, detection) tag (purification, detection) CDS CDS CDS CDS CDS regulation regulation CDS resistance cassette resistance gene tag (purification, detection) tag (purification, detection) codon polyA site replication origin resistance cassette Stuffer between CDS and terminator from Neomycin resistence cassette in pcDNA3.1 Construction Stuffer 26 GeneArt portal: Building&Engineering tab -> Elements vector construct 27 Elements: Enter 28 Elements: Edit 29 GeneArt® Elements™ Combinatorial Parts Assembly Combinatorial parts assembly (CPA) is a way to combine predefined DNA parts (e.g., promoters, ribosomal binding sites, open reading frames, terminators, etc.) in order to build a diverse set of larger constructs. Users simply select parts from a menu and send the request. The final construct is synthesized, with sequence junctions and frame all handled seamlessly by the assembly. All conceivable part combinations can be created to build and test new metabolic pathways or a variety of expression cassettes to identify the most valuable combination. • Tested for accuracy—All CPAs that are delivered as separate constructs are sequenced as part of our ISO 9001:2008–certified quality management system; we only ship constructs with exact sequence agreement • Cost-effective—As parts of the final constructs have the potential to be used multiple times • Comprehensive—All permutations of the available genetic elements are possible 30 GeneArt® Elements™ CPA: Application Example P xxx Firefly luciferase constitutive promoters P P P P P T yyy yeast terminators ADH1 T ADH1 CYC1 T CYC1 TEF1 T TEF1 URA3 HIS3 T T URA3 HIS1 31 Genome Editing Tools Precision TALs PerfectMatch TALs CRISPR Nuclease Vector Kits CRISPR Nuclease mRNA CRISPR U6/T7 Strings The world leader in serving science 32 Specificity TALs RNAi ZFNs CRISPR CRISPR ZFNs RNAi Manufacturing Complexity Genome Editing Tools TALs 33 Genome editing with engineered nucleases Random mutagenesis Targeted gene editing No Precision or control Precision and Control 34 TAL effector technology Precise and flexible editing The world leader in serving science 35 TAL effector technology • Transcriptional Activator –Like effectors from Xanthomonas and Ralstonia bacterial sp. • Found in bacterial strains that cause major crop diseases Plant cell • Pathogen proteins, up to 26 per strain • TALs secreted by Xanthomonas bacteria through their type III secretion system when they infect plants; this rewires host transcription and aids bacterial infection • DNA sequences are recognized through a central repeat domain Transcripts Bacteria Nucleus 36 Understanding the TAL code • Each repeat consists of 34 amino acid units that are nearly identical except for 2 hypervariable residues Repeat variable di-residue (RVD) • Each repeat contacts 1 DNA base pair; the 2 hypervariable residues determine nucleotide specificity • The nucleotide specificity of the variable amino acids has been decoded Boch et al. 2009, Science 37 Designer TAL effectors are vehicles to deliver genomic tools They can be coded to deliver functionality to specific loci: Nucleases Activators Repressors Chromatin modifiers Genomic labels SMA GFP TAATGCATGGCAA A T G C …and known to function in various hosts Bacteria Algae Yeast Plants Flies Zebrafish Mammals 38 Available effector domains Fok1 Nuclease Pair Nuclease function (Gene targeting via Fok1) Activator function • • • • Gene targeting Gene silencing Incorporation of exogenous DNA Targeted deletions • Gene activation • Gene expression Activator vp16 or vp64 (Activator vp16 or vp64) Repressor KRAB Repressor function (Epigenetic • Down-regulation gene expression (similar to the function of siRNA) • Heritable knock-down of gene expression repression via KRAB) MCS Vector Custom function (Custom design via MCS vector) • To target any locus in the genome with the effector domain of your choice– multiple cloning site vector • Transient knock-down of gene expression 39 TAL Assistant in the GeneArt portal www.lifetechnologies.com/genesynthesis 40 TAL Assistant 41 CRISPR-Cas9 genome editing Rapid and efficient editing with multiplexing capabilities The world leader in serving science 42 CRISPR/Cas 9 System • Background Information Prokaryotic adaptive immune system Uses RNA-guided DNA nuclease to silence viral nucleic acids The type II CRISPR/Cas system from Streptococcus pyogenes is a simple two component system CRISPR associated nuclease (Cas) non-coding guide RNA (gRNA) can be engineered to cleave genomic DNA at a predefined target sequence of interest. used in eukaryotic genome editing The gRNA has two molecular components CRISPR RNA (crRNA), complementary to the target an auxiliary trans-activating crRNA (tracrRNA) The gRNA unit guides the Cas9 nuclease to specific genomic locus where the Cas9 protein induces a double stranded break (DSB) 43 CRISPR/Cas9 System RNA-Guided DNA Nuclease System Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) Bacterial system with two components: a target specific guide RNA and a DNA cleaving nuclease, Cas9 Guide RNA (gRNA) = tracrRNA + crRNA chimera Cas9 nuclease has two catalytically active sites and can induce double stranded DNA break 44 Different CRISPR delivery formats 45 GeneArt® CRISPR Nuclease Vector Kits Target specific custom Oligo GeneArt® CRISPR Nuclease-CD4 vector 9822 bp CD4 Vector: For bead based enrichment of Cas9+gRNA expressing cells GeneArt® CRISPR Nuclease-OFP vector 9219 bp OFP Vector: For fluorescence based enrichment of Cas9+gRNA expressing cells 46 Flow based enrichment using OFP Vector Pre-sort: 9.5% of cells were OFP-positive Post-sort: 99 % of cells were OFP-positive Pre Pre Post Post Uncut Cut % Indel: 1.4 48.1 Flow-based analysis of OFP positive cells: for preand post- enrichment GeneArt® Cleavage Detection Assay: for pre- and post- enriched samples 47 Bead based enrichment using CD4 Vector Sample 1 Sample 2 Pre Flow-based analysis of FITC-stained CD4positive cells Post Sample 1 Sample 2 GeneArt® Cleavage Detection Assay: For pre- and post- enriched samples Uncut Cut 48 Unmet needs with vector based systems • Broad cell type or host application • Promoter constraints • Pay load size • Cas9 : gRNA ratio optimization • Multiplexing capability 49 GeneArt® CRISPR Nuclease mRNA (Cas9 mRNA) gRNA expression cassette with U6 polII promoter + or IVT gRNA generated from DNA template containing T7 promoter Advantages: • No promoter constraint for Cas9 expression • Smaller payload size • U6 CRISPR String™ =500 bp • IVT gRNA =100 bp • Allows Cas9 : gRNA dosage optimization • Amenable to multiplexing 50 Workflow: ready-to-transfect format Submit target sequence CRISPR design tool to design target-specific gRNA Synthetic gRNA cassette with Pol II promoter 51 Multiplexed gene editing with Cas9 mRNA + Genomic DNA cleavage efficiency 52 Workflow: complete RNA format gRNA as in vitro transcribed RNA Submit target sequence Target-specific gRNA design & synthesis 53 Cas9 mRNA + IVT gRNA for HPRT locus in iPS Cells 8.7% 0% Lipofectamine ® 3000 35.2% Lipofectamine ® MessengerMAX™ 54 Highly efficient multiplexed genome editing 55 Acknowledgements Synthetic Biology R&D Team Regensburg Synthetic Biology R&D Team Carlsbad MIT - Collaboration Dept Biological Engineering Chris Voigt, Ron Weiss Synthetic Biology Software Team Singapore 56 57
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