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Many New Applications for Synthetic DNA
AUGUST 7 , 2017 TW IST CONFIDENTIAL 4
Need a New Typeof DNA Supplier to
Meet Demand
Need a New Typeof DNA Supplier to
Meet Demand
CURRENT DNATECHNOLOGIES
RecombinantDNA
GenomicAdvances
Cloning
AgriculturalOil-Free Fertilizers
Drought Solutions
New Disease Protection
Biotech / PharmaAntibodies / TCR
Vaccines
Immuno and Cancer Therapies
Small Molecule Drug Mfg.
Industrial Bio-ProductionSpecialty Chemicals
Advanced Property Materials
Today’s Market: Buyers and Makers
5AUGUST 7 , 2017 TW IST CONFIDENTIAL
Small Scale, Academic Users
Price-Sensitive
Large Scale, Commercial Users
Value Speed, Throughput and
Quality
$1B / Year
“Buy”
Synthetic DNA
$250M
“Make”Synthesis Supplies
(Enzymes, Primers, Cells,
Plates)$750M
Can’t GetWhat I Need!
“ ”I Hate Cloning!
“ ”
Source: Percepta Associates
Current Technologies Are Limiting
AUGUST 7 , 2017 TW IST CONFIDENTIAL 6
PROCESS BOTTLENECK
• Too long
• Can’t scale up
or down
• Expensive
Select/Screen for Desired FunctionE.g., Bacteria, Yeast, Algae Bio-Factories, Plants,
Mammalian Cells
Design Gene Sequences1 to 10,000+ Variants Possible per Cycle
AGCTGCTCGAATACGATAG
Manufacture SyntheticGene Constructs• Genes (1k Basepairs)
• Parts (3 - 10 Genes)
• Pathways (10 - 100 Genes)
• Chassis (100 - 1000 Genes)
Build
Test
Cycles5-10x
Want 1 Gene in 2 Weeks $0.2- $1/bp or Non-Clonal + Work
Want 10,000 Genes in 2 Weeks Use 3 Vendors and/or Wait 3 Months
Limitations of 20-Year Old Technology
7
96 Well Plastic Plate
96 Oligos = 1 Gene
Does Not Scale to Meet Market
Need
Does Not Scale to Meet Market Need
Rewriting DNA Synthesis with the
Power of Silicon
AUGUST 7 , 2017 TW IST CONFIDENTIAL 8
Developing Game-Changing Throughput, Speed, Cost and Quality
96 Oligos 1 Gene
TRADIT IONAL METHODS
Based on 96-well plateTWIST BIOSCIENCE
Silicon-based Synthesis Platform
> 1 Million Oligos 9,600 Genes
3.2kb genes are at the core of various pipelines
Antibody-based
drug development
Up to 3.2 kb
Gene editing:
donor DNA synthesis
Photo sources:
medicalxpress.com, plos.org
Pathway assemblies
Gene therapy
10
Your Sequence,
Your Way
• Twist’s or your vector
• 100% NGS Verified
• up to 3.2kb
Industry Leading
Price & Fast
Turnaround Time
• 9c/bp
• 15-20 days TAT
Platform Scalability
• No order limits
• 1 – 10,000+ genes
Why Clone? Let Twist Build It
G E N E S
11
Your Sequence,
Your Way
• >1 error 3,000 bp
Industry Leading
Price & Fast
Turnaround Time
• 7c/bp
• 7 days TAT
Platform Scalability
• No order limits
• 1 – 10,000+ genes
Need Linear DNA? Let Twist Build It
N O N - C L O N A L F R A G M E N T S
Market Leader in Gene Synthesis
12
G E N E S P R O D U C E D A N D S H I P P E D
0
5,000
10,000
15,000
20,000
25,000
30,000
35,000
40,000
Jan-Mar 16 Apr-Jun 16 Jul-Sep 16 Oct-Dec 16 Jan-Mar 17
Shipped
Produced
Apr – Jun 2016: 2,000 genes per month
Jan – Mar 2017: 10,000 genes per month
December 2017: 60,000 genes per month
We Can Make
1 Million
Guide RNA Pairs
D E S I G N & B U I L D
P A R T N E R S H I P
Fueling Genome-Wide Experiments
13
C R I S P R - C A S 9 S Y S T E M
Novel Protein Libraries for Drug DiscoveryEnabling Efficiency in Drug Development
14
From Needlein a Haystack
• Random diversity
• Biased representation
• >99% inefficiency
• Lengthy optimization
cycle
• Expensive process
Gene Synthesis
Single Site
Multi-Site
Stretch
Multi-Domain
Precise Introduction of Variants,
Diversity that Enables Screening
Efficiency
.
. .
.
.
.
.
.
. .
Gene Synthesis
Single Site
Multi-site
Stretch
Multi-Domain
...
.
..
To Stack of Needles
• Explicit
• Even representation
• Human repertoire
based
• Fast
• Affordable
Protein Drug Discovery
½ the time
¼ the cost
15
P R E C I S I O N L I B R A R I E S
High Quality Precision
Libraries
• Single Site Variant Libraries
• Combinatorial Libraries
High Diversity
• 104 to 1010 variants
• 100% explicit sequence,
no random mutations
Industry Leading Price &
Rapid Turnaround Time
• ½ the time
• ¼ the cost
vs.
codon-by-codon
synthesis
52
1
GFPPX
Assay Development
Construct
mutagenesis library
Pharmacological
Characterization
Circuit integration
DNA Assembly ScreeningTransformation
Rational Design vs Random Mutagenesis
CH
AR
AC
TE
RIZ
AT
IO
N
Sequencing
PX
PX
Acknowledgements:
David Oling
Lina Lawenius
Niklas Larsson
Mark Wigglesworth
Tom Ellis
William Shaw
4
Random mutagenesis
(epPCR)
VS.
Saturation mutagenesis
Twist Saturation Mutagenesis Library
Highly uniform representation of every mutant
across all 161 positions
Less Work, More Hits
CriteriaTwist
Saturation libraryError prone PCR library
Variants All variants present Unknown
Number of Hits 10/10 hits2/10 (both present in Twist
library)
Overall
+ More hits
+ Pure, cloning ready DNA
+ Fast production time
- Less hits
- Unknown variants
- Large fraction of empty vectors
Finely Tuned Precision Libraries
Objective: Introduce variants at low frequency to mimic natural
distribution within the human immune repertoire
*Sequence and data anonymized
SPRIPWLCI??F?QVAE?LL??FEAHDPHPAACLFLIIPGH
Variable Positions 1 2 3 4 5 6
Total Diversity 1,391,208
Highly Precise Variant Introduction
Position 1
Expected Observed
A 2.7% 2.8%
C 8.6% 8.0%
D 2.7% 2.7%
E 8.6% 8.4%
F
G 1.7% 1.5%
H 8.6% 8.8%
I
K
L 8.6% 8.9%
M 1.7% 1.5%
N 1.7% 1.8%
P 2.7% 2.8%
Q 8.6% 7.6%
R 8.6% 7.9%
S 8.6% 8.3%
T 8.6% 9.0%
V
W
Y
0.00%
1.00%
2.00%
3.00%
4.00%
5.00%
6.00%
7.00%
8.00%
9.00%
10.00%
A C D E F G H I K L M N P Q R S T V W Y
Position 1Expected
Observed
Χ2 = 1
*Sequence and data anonymized
Highly Precise Variant Introduction
Position 2
Expected Observed
A 2.7% 2.9%
C 6.3% 6.6%
D 2.7% 2.7%
E 6.3% 6.2%
F
G 7.7% 7.4%
H 6.3% 6.3%
I
K
L 6.3% 6.4%
M 7.7% 7.0%
N 7.7% 8.5%
P 2.7% 2.8%
Q 6.3% 5.4%
R 6.3% 5.9%
S 6.3% 5.9%
T 6.3% 6.2%
V
W
Y
0.00%
1.00%
2.00%
3.00%
4.00%
5.00%
6.00%
7.00%
8.00%
9.00%
A C D E F G H I K L M N P Q R S T V W Y
Position 2Expected
Observed
Χ2 = 1
*Sequence and data anonymized