Advances within Gene Synthesis
and Genome Editing
Anja Martinez, PhD
Technical Sales Specialist – Synthetic Biology
Biosciences, Life Sciences Solutions,
Geneart AG, Regensburg, Germany
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Agenda
• Gene synthesis
• Optimization
• Strings DNA Fragments
• Directed evolution
• Elements
• Vector construction
• Combinatorial parts assembly
• Genome editing
• Introduction
• Tools
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Gene synthesis
Gene synthesis
Optimization
Strings DNA Fragments
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GeneArt – Synthetic genes
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• 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
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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
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GeneArt GeneOptimizer
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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
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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
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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.
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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
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Online ordering portal
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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
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GeneArt® Strings™ DNA Fragments
Your source for genes.
Fast and affordable for every lab.
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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
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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)
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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
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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
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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!
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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
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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
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Rational Directed Evolution Workflow
Short cuts:
• Structural data
• Homology data
• Literature
• etc
Screen
Improved variant
Wild type
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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
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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.
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GeneArt® ElementsTM Vector Construction
and Combinatorial Parts Assembly
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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
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GeneArt portal:
Building&Engineering tab -> Elements vector construct
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Elements: Enter
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Elements: Edit
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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
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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
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Genome Editing Tools
Precision TALs
PerfectMatch TALs
CRISPR Nuclease Vector Kits
CRISPR Nuclease mRNA
CRISPR U6/T7 Strings
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Specificity
TALs
RNAi
ZFNs
CRISPR
CRISPR
ZFNs
RNAi
Manufacturing Complexity
Genome Editing Tools
TALs
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Genome editing with engineered nucleases
Random mutagenesis
Targeted gene editing
No Precision or control
Precision and Control
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TAL effector technology
Precise and flexible editing
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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
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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
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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
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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
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TAL Assistant in the GeneArt portal
www.lifetechnologies.com/genesynthesis
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TAL Assistant
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CRISPR-Cas9 genome editing
Rapid and efficient editing with multiplexing capabilities
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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)
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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
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Different CRISPR delivery formats
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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
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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
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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
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Unmet needs with vector based systems
• Broad cell type or host
application
• Promoter constraints
• Pay load size
• Cas9 : gRNA ratio optimization
• Multiplexing capability
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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
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Workflow: ready-to-transfect format
Submit target sequence
CRISPR design tool to design target-specific gRNA
Synthetic gRNA cassette with Pol II promoter
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Multiplexed gene editing with Cas9 mRNA
+
Genomic DNA cleavage efficiency
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Workflow: complete RNA format
gRNA as in vitro transcribed RNA
Submit target sequence
Target-specific gRNA design & synthesis
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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
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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
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