diy gene variant libraries - agilent...screening costs to quikchange ht 3.4 11.3 38.2 128.7 433.6...

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

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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

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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

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

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

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

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


G5901A G5901B

Commercial

10sites(1-500 AA)

20sites(500-1000 AA)


G5902A G5902B


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)

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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


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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


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


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


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The rational approach to mutagenesis according to the functional assay

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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


500-10,000 100-2,000QuikChange HT Codon Saturation scanning entire

domain/protein or multiple proteins

Error Prone PCR


10,000-500,0002,000-120,000

QuikChange HT

1. Codon Saturation Scanning (entire protein)

2. Targeted Combinatorial Libraries

Error Prone PCR


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


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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


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?


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?


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


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


diy gene variant libraries - agilent...screening costs to quikchange ht 3.4 11.3 38.2 128.7 433.6...

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