DIY gene variant libraries
QuikChange HT Protein Engineering System Agilent’s Contribution in Synthetic Biology
• Breadth of Portfolio - Agilent has platforms across its businesses that can address much of the synthetic biology needs - Agilent offers complete workflow solutions through new product introduction and product enhancement • Leverage Core Technologies - Microarray fabrication facility is a key competence – OLS oligo libraries of high quality and complexity - Incorporation of OLS oligo libraries into high value tools including CRISPR, Advanced Cloning and Protein Engineering platforms • Partner with Gen9 - Combining forces, Agilent and Gen9 offer researchers more options for their experiments than previously available. - Agilent OLS enables Gen9 to manufacture long, accurate genetic constructs more efficiently. Applications for OLS libraries
Gene and genome Selective target Probes for in situ High throughput site synthesis enrichment for high hybridizations directed mutagenesis Next Generations Sequencing (NGS)
GEN9 • SureSelect SureFISH QuikChange HT • Halo Site-Directed Mutagenesis Applications
Structure/Function Studies
• Protein Folding • ID functional domains, phosphorylation sites • Protein/Protein Interactions Studies Limited By: • Cost of comprehensive Protein Engineering mutational strategies • Lack of structural • Increased immunogenicity information • Reduced toxicity • Screening capabilities • Codon optimization • Increased enzyme activity
Correcting Unwanted Mutations
• Cloning • Gene Synthesis
Agilent Confidential May 21, 2015 4 Mutagenesis Approaches for Increased Coverage
Random +Wide range, straight forward and divergent mutagenesis -Codon bias, low coverage, multisite, requires validation, more (e.g. Error- screening Prone PCR)
Site-Directed +Targeted, Conclusive, Indels Mutagenesis -Targeted, multiple reactions, costly > Wait Structural Information
Gene Variant +Most Informative, rational design libraries -Long lead time, costly, not kitted
Libraries +Comprehensive coverage, reduced synthesis cost with degenerate -Codon bias, exponential screening costs content with number of degenerated sites in combinatorial assays
Agilent Confidential May 21, 2015 5 Microarray based Oligo libraries for SDM
Mutagenesis OLS libraries: Up to 120,000 user- synthetic DNA definable sequences /chip
Due to the limited amount synthesized on single features, libraries need to amplified prior to use • Libraries are provided as ssDNA 20-25 bps 150-160 bps 20-25 bps • Several sub-libraries can be printed on the same chip • PCR acts as clean-up step • Control features are integrated into the 200 mer library design Chemical Synthesis: Achieving High synthesis efficiency Depurination Long length synthesis is achieved 1) Coupling side reaction by improved cycle yield •↑ coupling efficiency •↓ depurination Inkjet •↑ consistency
99.8% cycle yield 3) Deblock 2) Oxidation Flood
Repeat n times N HO O i O N O P O O 2 RO 150mer complex library O N O P O O 1 RO PCR O O P O RO QuikChange HT approach to protein engineering 15-50aa Rational HT mutagenesis
• Up to 120K total variants • Up to 4 codons mutated per oligo • 10-20 oligo sets15-50aa to cover up to 1000aa SurePrint Oligo Library Synthesis 1. Synthetic mutagenic • 100-200mers DNA oligo library • Two adapter/priming regions Oligo Oligo set 1 set 2 (2x25bases) • Up to120,000 variants
2 . Amplify and Purify • 1,2,3…20 oligo sets each oligo sets separately • Up to 50 codons/oligo set (sub-libraries) • pfu Ultra II HS polymerase • PCR Controls
3. Mutant Strand Synthesis • Linear amplification with GOI • Denature DNA template QuikChange lightning fusion • Anneal Mutagenic Oligo Set polymerase >Maximum fidelity • Extend and Integrate
Each oligo set a separate transformation reaction
4. Digest parental methylated and hemi- • 5 min digestion methylated DNA • Optimized Dpn I formulation
5. Transformation • SoloPack Gold Supercompetent Cells
(resistant to tetracycline and chloramphenicol)
Agilent Confidential May 21, 2015 9 QuikChange HT - Workflow
QC HT Step 1 : Design mutagenic library specific to QC HT Step 2: Library is constructed per design, oligo sets are tagged with specific sequence and application with assay design software. primer sequences for PCR amplification and purification from the total library
SurePrint Inkjet Oligo Oligo library Library Synthesis cleaved eArray Design a) Single AA scanning c) Combinatorial mutagenesis PCR & Purify b) Codon saturation scanning oligo sets 1,2,3...20
E.coli library QC HT Step 3: Perform QuikChange mutagenesis with each oligo set, move onto mutant screening in less than 24 hours
Screen – Sequencing Site Directed Mutagenesis Identify and Sequence distinct clones Incorporate oligo sets separately into plasmid DNA with QuikChange, followed by Dpn I enrichment & transformation
Page 10 Codon saturation scanning of GFP: the set-up
chromphore E120G
Master library Sub-libraries GFPOLS1 GFPOLS2 GFPOLS3 Oligo length 189-200 bases 200 bases 189 bases 198 bases Mutation type “QuikScan-19” codon saturation codon saturation codon saturation Mutation region 3 x 50aa regions 28-77 (chromophore) 104-153 181-230 Expression host E. coli E. coli E. coli E.coli # unique sequences 5701 b 1900 a 1900 a 1900 a
° Gene target: humanized Renilla reniformis GFP (hrGFP) ° Library: One custom library targeting three 50-amino acid domains (60% of total protein) ▫ designed using QuikChange HT mutagenesis primer design software in eArray ° Mutagenesis strategy: “QuikScan-19” (Site saturation mutagenesis of 3 x 50 = 150 codons) ° Screen: increased fluorescence in E. coli ° Evaluation: Compare to previous results obtained using random mutagenesis (whole 720bp gene) ▫ isolated brighter hrGFP mutant (E120G; GAG→GGG) Brighter than hrGFP
Location of brighter mutations: brighter than hrGFP; brighter than hrGFP II (E120G; )
Page 12 Codon saturation scanning of GFP: sub -library screening
GFP-OLS1 GFP-OLS2 GFP-OLS3
Length (bases) 200 189 198
N-terminal Mutation region (amino acids) middle C-terminal (chromophore)
QuikChange library size (x10 4) 7.8 14 8.9
% fluorescent clones 0.5 5.9 7.7
# colonies screened (x10 4) 1.32 2.5 1.54
49 (1) – 10 (2) 186 (1) – 38 (2) # positives isolated (per round) 360 (1)-17(2) – 4(3) – 10(3) # positives brighter than 1/0 14/8 10/7 hrGFP/hrGFP II ( E. coli )* # brighter with secondary amino 4 double 1 double and 1 0 acid changes mutants triple mutant
Page 13 Comparison of mutations isolated by different techniques*
Mutagenesis Method (Primary library size) Mutations that increase fluorescence Strategy (E. coli) Number Screened Confirmed Putative (Single) (Multiple) EP-PCR Mutazyme (4 libraries: F43, L101 R102, E120 , R125 , (whole gene) (GeneMorph kit) 2.3 x 10 4-2.8 x 10 5) V215, K230 NR Taq/Mn 2+ (NR) Y103, E120 M16, N21, T32, F43, V109, V123 , K142, NR S173, T207, F214, V215 Codon QuikScan-19 (5700) N28, N116, E120 , R125 , L129, M185, saturation M121, V123 , Y124, G229, G233 5.4 x 10 4 (60% gene) R131, V154, L184, F194, G213 *(underline) mutations isolated by EP-PCR that fall outside of QuikScan target region; NR, not recorded
EP-PCR identified significantly fewer single mutants than QuikChange HT
QuikChange HT Site Saturation Scanning: ° identified same site as EP-PCR (E120, but mutation is D instead of G) ° identified different mutation sites (only 3 common to both techniques) ° produced 7 independent mutations that are brighter than E120G isolated by EP-PCR
Page 14 Ordering options
Commercial Academic/Government/NGO 10sites 20sites 10sites 20sites (1-500 AA) (500-1000 AA) (1-500 AA) (500-1000 AA) 150nt G5900A G5900B 150nt G5902A G5902B (1-33AA/region) (1-33AA/region) 200nt G5901A G5901B 200nt G5903A G5903B (33-50AA/region) (33-50AA/region) Pricing based on target region size, not number of mutants (coverage)
Lowest cost per transformed rationally designed mutant All Inclusive: Mutagenic Primer Library + Library Amplification and Cleanup + + QuikChange Lightning + + Competent Cells + Controls and the fastest protocol
1. QuikChange HT Mutagenesis Library (one tube) 2. QuikChange HT Mutagenesis Sub-Library Primers (2-40 tubes) 3. QuikChange HT Mutagenesis Reagents 4. QuikChange HT DNA Cleanup Kit Room Temperature (bind & wash buffers + spin cups) 5. SoloPack Gold Supercompetent Cells (15 tubes + 1pUC18)
15 Relative Clone Screening Costs by Relative Coverage of the mutagenesis alternative techniques space by alternative techniques
P EP-PCR dS
Degenerate Synthetic High Q H dS Gene Variant Libraries
Random M S D SDM+Degenerate Oligos (QuikChange Multi+Degen. O.) Relative coverage
H QuikChange HT: Medium SDM+OLS D Targeted Synthetic S Rational Variant Libraries P
M SDM – Multi (i.e. QuikChange Low Lightning Multi) 0 SDM (i.e. QuikChange 1 10 10 2 10 3 10 4 Screening Screening Costs Q Lightning) target size (bp)
• Screening costs for random and degenerate libraries increase • Relative Screening requirements to find a low abundant mutant exponentially for combinatorial libraries indicated by fill in color intensity QuikChange HT provides the best value: • Lowest cost/mutant • Information Rich: Wide and comprehensive region coverage • Efficient: Rational design minimizes screening costs
Agilent Confidential May 21, 2015 16 Lowest cost per transformed mutant Mutagenic Primer library + QuikChange + Competent Cells All-in-One Whole protein Single Amino Acid scanning • Identify functional regions of uncharacterized proteins Whole protein Codon Saturation Scanning • allows precise mapping of functional features at the atomic level Targeted combinatorial mutagenesis • Rationally design combinations • Quick optimization of specified combinations (protein expression & activity) Codon optimization Target multiple entire domains in one or more proteins
Agilent Confidential May 21, 2015 17
Increasing screening costs when using Combinatorial libraries with degenerate content
QuikChange HT Rational designed libraries* AA positions 1 2 3 4 56 7 8 9 10 codons 19 19 19 19 19 19 19 19 19 19 effective combinations 19 361 6859 1.30E+05 2.48E+06 4.70E+07 8.94E+08 1.70E+10 3.23E+11 6.13E+12 NNK Degenerate libraries AA positions 1 2 3 4 5 6 7 8 9 10 codons 32 32 32 32 32 32 32 32 32 32 effective combinations 32 1024 32768 1.05E+06 3.36E+07 1.07E+09 3.44E+10 1.10E+12 3.52E+13 1.13E+15 Screening costs to QuikChange HT 1.7 2.8 4.8 8.0 13.6 22.8 38.4 64.7 109.0 183.6 NNN Degenerate libraries AA positions 1 2 3 4 5 6 7 8 9 10 codons 64 64 64 64 64 64 64 64 64 64 effective combinations 64 4096 262144 1.68E+07 1.07E+09 6.87E+10 4.40E+12 2.81E+14 1.80E+16 1.15E+18 Screening costs to QuikChange HT 3.4 11.3 38.2 128.7 433.6 1460.7 4920.2 16573.4 55826.1 188045.8 *Only combinations up to 4sites are currently offered
Agilent Confidential May 21, 2015 19 The rational approach to mutagenesis according to the functional assay
Screening Number of variants in library capacity with Mutagenesis Product Recommended Mutagenesis (95% variant representation in Mutagenesis Approach replaced Functional approach screen) Assay (clones)
1. QuikChange Lightning Site Directed Mutagenesis 3-100 1-30 2. QuikChange Lightning PCR SDM Single or MultiSite Multi
QuikChange HT Single AA scanning of the entire Error Prone PCR domain/protein Synthetic Gene Variant libraries
100-1,000 30-250 QuikChange Lightning Multi Targeted codon saturation at up to 5 Error Prone PCR + Degenerate Oligos individual sites Synthetic Gene Variant libraries
QuikChange HT Codon Saturation scanning entire Error Prone PCR 500-10,000 100-2,000 domain/protein or multiple proteins Synthetic Gene Variant libraries
1. Codon Saturation Error Prone PCR 10,000-500,000 QuikChange HT Scanning (entire protein) 2,000-120,000 Synthetic Gene Variant libraries 2. Targeted Combinatorial Libraries Gene Variant libraries with degenerate Degenerate Variant content for complex combinatorial Error Prone PCR 10 6-10 10 10 5-10 9 libraries* libraries
*Increasing screening costs when using combinatorial libraries with degenerate content (1.7x for a single degenerate site, and up to 8x for combinations of 4 sites)
Agilent Confidential May 21, 2015 20 CRISPR Tools for the Age of Synthetic Biology
SureGuide Fast, Flexible, and Easy: Purified Cas9 + gRNA synthesis system All-in-One Fully Customizable Nuclease Specificity • Harness the power of the next-generation of genome editing tools • Target any region of interest, including long, complex stretches of DNA Synthesize gRNA on demand • High quality, high yield IVT kit assures reliable results • Fast, simple, and easy to obtain new guides for different applications Simple, validated reagents you can be Sure of • Faster start-up times with validated and purified reagents • Optimized systems allows you to focus on the science, not the tools
Agilent Confidential May 21, 2015 22 The CAS9/CRISPR system targets double stranded
DNA for cleavage dispensable tracrRNA CAS9 CAS2 CAS1 csn1 CRISPR
Repeat sequence spacer Repeat sequence TACGAGGTTTTAGAGCTGTGTTGTTTCGAATGGTTCCAAAAC AGTAATATCAAAAAAGCCCCCTTGATTATC GTTTTAGAGCTGTGTTGTTTCGAATGGTTCCAAAAC For a 50% GC genome PAM Cleavage site PAM site sites are expected to occur Genomic DNA ..NNNNNNNN AGTAATATCAAAAAAGCCCCCTTGATTATC NGG NGNNN…… 1/32 base pairs
crRNA processing tracrRNA and crRNA can be linked to mimic Spacer processed form (guide sequence) crRNA RNAse III
tracrRNA
Agilent confidential in vitro DNA cleavage by CAS9
Needed for in-vitro cleavage
CAS9
CAS9
guide RNA (sgRNA)
spacer (20 nt) stem (30 nt)
tracr tail (13 nt)
OH ? PAM 5’ 3’ 3’ 5’ ? OH OH ? 5’ 3’ 3’ 5’ ? OH
Agilent confidential What are the current primary uses of CAS9?
requires NLS on CAS9! CAS9 CAS9 expression vector
CAS9 CAS9
Purified CAS9 protein Guide RNA expression vector NHEJ purified guide RNA • Leads to scar at cleavage site (indels) • mutates target site
Libraries of guides Homologous recombination DNA oligo libraries Libraries of inserts
targeted, concise insertion or replacement
Agilent confidential Are cleavage products compatible with down stream molecular biology applications?
Cloning of 156 bps fragment
10 randomly picked clones analyzed by restriction analysis
Expected product sizes: 3.9 kB, 180 bps
All clones were the expected product
Agilent confidential Cloning of CAS9-digested DNA fragments
Cloned CAS9-excised 5.4 kb fragment into Strataclone blunt vector
Selected for marker (gentamycin resistance) encoded by cloned fragment
Picked 10 colonies for restriction analysis
Analyzed cloning junctions of 5 isolates
Additional nucleotide at junction is due to cleavage at the -4 position instead of the -3 position relative to the PAM site
Wobble cleavage occurs at a frequency of ≈1/20
Agilent confidential adaptors can be ligated to CAS9 digested sites
CAS9
cleave target with CAS9
P noCAS9 CAS9digested P ligate adaptors with T4 DNA ligase
purifiy ligation product
PCR amplify ligation product gRNA libraries: Functional Genomics
gRNA Oligo Array Synthesis gRNAs cleaved & Cloning & Purification PCR amplified
Viral packaging Pathway analysis Treatment (e.g. drug resistance vs. Control KO assay)
Screening and Transduction Hit sequencing and selection of Target Cells SureGuide Ordering
Agilent Confidential