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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
GEN9 SureFISH QuikChange HT• SureSelect
• Halo
Gene and genome
synthesis
Selective target
enrichment for high
Next Generations
Sequencing (NGS)
Probes for in situ
hybridizations
High throughput site
directed mutagenesis
Site-Directed Mutagenesis Applications
May 21, 2015
Agilent Confidential
4
• Protein Folding
• ID functional domains, phosphorylation sites
• Protein/Protein Interactions
Structure/Function Studies
• Increased immunogenicity
• Reduced toxicity
• Codon optimization
• Increased enzyme activity
Protein Engineering
• Cloning
• Gene Synthesis
Correcting Unwanted Mutations
Studies Limited By:• Cost of comprehensive
mutational strategies
• Lack of structural
information
• Screening capabilities
Mutagenesis Approaches for Increased Coverage
May 21, 2015
Agilent Confidential
5
Random mutagenesis
(e.g. Error-Prone PCR)
+Wide range, straight forward and divergent
-Codon bias, low coverage, multisite, requires validation, more screening
Site-Directed Mutagenesis
+Targeted, Conclusive, Indels
-Targeted, multiple reactions, costly
> Wait Structural Information
Gene Variant libraries
+Most Informative, rational design
-Long lead time, costly, not kitted
Libraries with degenerate content
+Comprehensive coverage, reduced synthesis cost
-Codon bias, exponential screening costs with number of degenerated sites in combinatorial assays
synthetic DNA
Microarray based Oligo libraries for SDM
Mutagenesis OLS
libraries:
Up to 120,000 user-
definable sequences
/chip
Due to the limited amount synthesized on single features, libraries need to amplified prior to use
200 mer
150-160 bps 20-25 bps20-25 bps
• Libraries are provided as ssDNA
• Several sub-libraries can be printed on the
same chip
• PCR acts as clean-up step
• Control features are integrated into the
library design
Chemical Synthesis: Achieving High synthesis efficiency
Long length synthesis is achieved
by improved cycle yield
•↑ coupling efficiency
•↓ depurination
•↑ consistency
3) Deblock
1) Coupling
2) Oxidation
Repeat n times
Depurination
side reaction
N1
N2
NiO
OP O
ROO
HO
InkjetInkjet
FloodFlood
O
O
O
O
P ORO
O
P ORO
O
PCR
150mer complex library
99.8% cycle yield
15-50aa
QuikChange HT approach to protein engineeringRational HT mutagenesis
• Up to 120K total variants
• Up to 4 codons mutated
per oligo
• 10-20 oligo sets15-50aa
to cover up to 1000aa
1. Synthetic mutagenic
DNA oligo library
• 100-200mers
• Two adapter/priming regions
(2x25bases)
• Up to120,000 variants
2 . Amplify and Purify
each oligo sets separately
(sub-libraries)
• 1,2,3…20 oligo sets
• Up to 50 codons/oligo set
• pfuUltra II HS polymerase
• PCR Controls
3. Mutant Strand Synthesis
• Denature DNA template
• Anneal Mutagenic Oligo Set
• Extend and Integrate
Each oligo set a separate
transformation reaction
• Linear amplification with
QuikChange lightning fusion
polymerase >Maximum fidelity
4. Digest parental methylated and hemi-
methylated DNA
• 5 min digestion
• Optimized Dpn I formulation
5. Transformation • SoloPack Gold Supercompetent Cells
(resistant to tetracycline and
chloramphenicol)
SurePrint Oligo Library Synthesis
Oligoset 1
Oligoset 2
GOI
May 21, 2015
Agilent Confidential
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PCR & Purify oligo sets 1,2,3...20
SurePrint Inkjet Oligo Library Synthesis
QC HT Step 2: Library is constructed per design, oligo sets are tagged with specific
primer sequences for PCR amplification and purification from the total library
Oligo librarycleaved
QuikChange HT - Workflow
QC HT Step 1: Design mutagenic library specific to
sequence and application with assay design software.
QC HT Step 3: Perform QuikChange mutagenesis
with each oligo set, move onto mutant screening in
less than 24 hours
Site Directed MutagenesisIncorporate oligo sets separately into plasmid DNA with
QuikChange, followed by Dpn I enrichment & transformation
E.coli library
Screen – Sequencing Identify and Sequence distinct clones
eArray Designa) Single AA scanning c) Combinatorial mutagenesisb) Codon saturation scanning
Page 10
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 5701b 1900a 1900a 1900a
Codon saturation scanning of GFP: the set-up
chromphore
� 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)
E120G
Location of brighter mutations: brighter than hrGFP; brighter than hrGFP II (E120G; )
Brighter than hrGFP
Page 12
GFP-OLS1 GFP-OLS2 GFP-OLS3
Length (bases) 200 189 198
Mutation region (amino acids) N-terminal
(chromophore)middle C-terminal
QuikChange library size (x104) 7.8 14 8.9
% fluorescent clones 0.5 5.9 7.7
# colonies screened (x104) 1.32 2.5 1.54
# positives isolated (per round)49 (1) – 10 (2)
– 4(3)360 (1)-17(2)
186 (1) – 38 (2)
– 10(3)
# positives brighter than
hrGFP/hrGFP II (E. coli)*1/0 14/8 10/7
# brighter with secondary amino
acid changes0
4 double
mutants
1 double and 1
triple mutant
Codon saturation scanning of GFP: sub-library screening
Page 13
Comparison of mutations isolated by different techniques*
Mutagenesis
Strategy
Method (Primary library size)
Number Screened
Mutations that increase fluorescence
(E. coli)
Confirmed
(Single)
Putative
(Multiple)
EP-PCR
(whole gene)
Mutazyme
(GeneMorph kit)
(4 libraries:
2.3 x 104-2.8 x 105)
NR
F43, L101 R102, E120, R125,
V215, K230
Taq/Mn2+ (NR)
NR
Y103, E120 M16, N21, T32, F43,
V109, V123, K142,
S173, T207, F214,
V215
Codon
saturation
(60% gene)
QuikScan-19 (5700)
5.4 x 104
N28, N116, E120,
M121, V123, Y124,
R131, V154, L184,
F194, G213
R125, L129, M185,
G229, G233
Page 14
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
*(underline) mutations isolated by EP-PCR that fall outside of QuikScan target region; NR, not recorded
Ordering options
15
10sites(1-500 AA)
20sites(500-1000 AA)
150nt(1-33AA/region)
G5900A G5900B
200nt(33-50AA/region)
G5901A G5901B
Commercial
10sites(1-500 AA)
20sites(500-1000 AA)
150nt(1-33AA/region)
G5902A G5902B
200nt(33-50AA/region)
G5903A G5903B
Academic/Government/NGO
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)
Pricing based on target region size, not number of mutants (coverage)
May 21, 2015
Agilent Confidential
16
Relative Coverage of the mutagenesis space by alternative techniques
target size (bp)
1 10 102 103 104
• Relative Screening requirements to find a low abundant mutant
indicated by fill in color intensity
0
Re
lative
co
ve
rage
High
Medium
Low
Q
M
D
S
H
P EP-PCR
QuikChange HT: SDM+OLS
Targeted Synthetic Variant Libraries
SDM+Degenerate Oligos(QuikChange Multi+Degen. O.)
SDM – Multi (i.e. QuikChange
Lightning Multi)
SDM (i.e. QuikChange
Lightning)
P
D
M
Q
S
H
Ra
tio
na
lR
an
do
m
Degenerate Synthetic Gene Variant LibrariesdS
dS
Relative Clone Screening Costs by alternative techniques
• Screening costs for random and degenerate libraries increase
exponentially for combinatorial libraries
Scre
enin
g C
osts
QuikChange HT provides the best value:
• Lowest cost/mutant
• Information Rich: Wide and comprehensive region coverage
• Efficient: Rational design minimizes screening costs
May 21, 2015
Agilent Confidential
17
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
Lowest cost per transformed mutant
Mutagenic Primer library + QuikChange + Competent Cells
All-in-One
QuikChange HT Rational designed libraries*
AA positions 1 2 3 4 5 6 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
Increasing screening costs when using Combinatorial libraries with degenerate content
May 21, 2015
Agilent Confidential
19
The rational approach to mutagenesis according to the functional assay
May 21, 2015
Agilent Confidential
20
Screening capacity with Functional Assay (clones)
Number of variants in library (95% variant representation in screen)
Mutagenesis Product Recommended Mutagenesis approach
Mutagenesis Approach replaced
3-100 1-30
1. QuikChange Lightning
2. QuikChange Lightning
Multi
Site Directed Mutagenesis
Single or MultiSitePCR SDM
100-1,000 30-250
QuikChange HTSingle AA scanning of the entire domain/protein
Error Prone PCR
Synthetic Gene Variant libraries
QuikChange Lightning Multi
+ Degenerate Oligos
Targeted codon saturation at up to 5 individual sites
Error Prone PCR
Synthetic Gene Variant libraries
500-10,000 100-2,000QuikChange HT Codon Saturation scanning entire
domain/protein or multiple proteins
Error Prone PCR
Synthetic Gene Variant libraries
10,000-500,0002,000-120,000
QuikChange HT
1. Codon Saturation Scanning (entire protein)
2. Targeted Combinatorial Libraries
Error Prone PCR
Synthetic Gene Variant libraries
106-1010 105-109Degenerate Variant
libraries*
Gene Variant libraries with degenerate
content for complex combinatorial
libraries
Error Prone PCR
*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)
CRISPR Tools for the Age of Synthetic Biology
SureGuide
May 21, 2015
Agilent Confidential
22
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
Fast, Flexible, and Easy:
Purified Cas9 + gRNA synthesis system
All-in-One
crRNAcrRNA
Spacer(guide sequence)Spacer(guide sequence)
crRNA processingcrRNA processing
crRNA
Spacer(guide sequence)
crRNA processing
CAS9CAS9 CAS2CAS2 CAS1CAS1csn1csn1 CRISPRCRISPRCAS9 CAS2 CAS1csn1 CRISPR
dispensable
tracrRNA
The CAS9/CRISPR system targets double stranded DNA for cleavage
Agilent confidential
tracrRNA
..NNNNNNNNAGTAATATCAAAAAAGCCCCCTTGATTATCNGGNGNNN……
Repeat sequence Repeat sequencespacer
Genomic DNA
Cleavage site
TACGAGGTTTTAGAGCTGTGTTGTTTCGAATGGTTCCAAAACAGTAATATCAAAAAAGCCCCCTTGATTATCGTTTTAGAGCTGTGTTGTTTCGAATGGTTCCAAAAC
PAM site
tracrRNA and crRNA can be linked to mimic processed form
For a 50% GC genome PAM sites are expected to occur 1/32 base pairs
For a 50% GC genome PAM sites are expected to occur 1/32 base pairs
RNAse III
in vitro DNA cleavage by CAS9
Agilent confidential
stem (30 nt)spacer (20 nt)
tracr tail (13 nt)
CAS9
Needed for in-vitro cleavage
guide RNA (sgRNA)
CAS9
5’
5’3’
3’
OH
OH
?
?
5’
5’3’
3’OH
OH
?
?
PAM
What are the current primary uses of CAS9?
Agilent confidential
CAS9
CAS9
Homologous recombination
targeted, concise insertion or replacement
CAS9 expression vector
Guide RNA expression vector
CAS9
Purified CAS9 protein
purified guide RNA
requires NLS on CAS9!
NHEJ
• Leads to scar at cleavage site (indels)• mutates target site
DNA oligo libraries
Libraries of guides
Libraries of inserts
Are cleavage products compatible with down stream molecular biology applications?
Agilent confidential
Expected product sizes:3.9 kB, 180 bps
Cloning of 156 bps fragment
10 randomly picked clones analyzed by restriction analysis
All clones were the expected product
Cloning of CAS9-digested DNA fragments
Agilent confidential
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
adaptors can be ligated to CAS9 digested sites
CAS9
PP
cleave target with CAS9
ligate adaptors with T4 DNA ligase
purifiy ligation product
PCR amplify ligation product
no
CA
S9
CA
S9
dig
este
d
gRNA libraries: Functional Genomics
gRNA Oligo Array Synthesis gRNAs cleaved & PCR amplified
Cloning & Purification
Viral packaging
Transductionand selection of Target Cells
Treatmentvs. Control
Screening and Hit sequencing
Pathway analysis (e.g. drug resistance
KO assay)
SureGuide Ordering
Agilent Confidential