Synthetic Biology: Writing & Editing DNA€¦ · Synthetic Biology: Writing & Editing DNA. 2 Proprietary & Confidential Breeding Accelerated breeding Biotechnology ... TTA L ATC ATT

The world leader in serving scienceProprietary & Confidential

2015-12-07 Korbinian Heil

Synthetic Biology: Writing & Editing DNA

2 Proprietary & Confidential

BreedingAccelerated

breeding

Biotechnology

colchizine

The biotechnological evolution


Vision for SynBio: Engineering Cycle for Biology

Characterize

Validate

Model

Engineer / Build

Rational Design

Target

Design


Focus Area for SynBio Research

Design

Build

Genome

Gene

Transcript

Protein

Cell

Systems

Phenome

Test

DNA manipulation as focus for SynBio:

�We build & modify biosystems by changing their information (i.e. DNA)

� Tools at gene level:• DNA-synthesis• Genome editing

SynBio

5 Proprietary & Confidential The world leader in serving scienceProprietary & Confidential

Research Tools from SynBio:Writing DNA


1975 1980 1985 1990 1995 2000 2005 2010

1 Tbp

1 Gbp

1 Mbp

1 kbp

automated dye terminator sequencing

manualradioactivesequencing

next generation sequencing

Exponential sequence data growth


1975 1980 1985 1990 1995 2000 2005 2010

1 Tbp

1 Gbp

1 Mbp

1 kbp

automated dye terminator sequencing

manualradioactivesequencing

next generation sequencing

hard

dis

k ca

paci

ty (

GB

)

Exponential sequence data growth ... follows Moore’s Law


This is a vast amount of data


ATG ATC TGT CAC GCA GAG CTA

...which we can read

...copy &paste

...and are ableto rewrite

This is a vast amount of data

comparecomparecomparecompare thee to a summer ?Shall I DAYDAYDAYDAY's


1.1

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Gib

son:

M. m

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p K

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eron

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kb

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: pol

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rus

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tem

mer

: pla

smid

77 b

p A

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al: a

la tR

NA

12 M

b S

c 2.

0

inventionof PCR

introduction of

commercial gene

synthesis

1970 1975 1980 1985 1990 1995 2000 2005 2010 2015

100,000,000

10,000,000

1,000,000

100,000

10,000

1,000

100

10

publ

iche

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[bp] 27

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Ann

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III

History of Writing DNA


1999: Foundaon → GeneArt GmbH� 3 employees

2006: Going public → GeneArt AG� 60 employees

2011: GeneArt a part of...� 200 employees (global 11,000)

2014: Life Technologies a brand of...� 270 employees (global 50,000)

1999 today

0.5genes / month

> 6000genes / month

The history of GeneArt® gene synthesis


Premier Brands

Global Scale • 50,000 employees in 50 countries

• $17 billion in annual revenues

• Unparalleled commercial reach

• 10,000 employees working with

customers every day

The World Leader in Serving Science

Unmatched Depth• Innovative technologies

• Applications expertise

• Lab productivity partner


Traditional cloning


� Save time and focus on your research

Traditional cloning vs. GeneArt® gene synthesis


• Synthetic Genes are double stranded DNA constructs, synthesized to the customer specification based on customers digital sequence

• Synthetic Genes can routinely be made > 10 kb in length

• Genes are delivered in a GeneArt® standard cloning vector or the vector of the customer’s choice

• Standard deliverable is 5 µg lyophilized DNA, larger amounts are available based on additional plasmid preparation

• All genes are 100 % sequence verified prior to shipment and come with quality assurance documentation

What are synthetic genes?


ATGAGTAAAGGAGAAGAACTTTTCACTGGAGTTGTCCCAATTCTTGTTGAATTAGATGGCGATGTTAATGGG

ATGAG AA GGGA GA CT TTCACTGG GTTGTCC ATTCT GTGA GA GGCGA GT AA GG

ATGAGCAAGGGCGAGGAGCTGTTCACTGGCGTTGTGCCCATTCTGGTGGAGCTGGACGGCGACGTGAACGGC

How gene synthesis works


ATGAGCAAGGGCGAGGAG

ATGAGCAAGGGCGAGGAG

ATGAGCAAGGGCGAGGAG

ACGT







colony





Pagothenia

borchgrevinki

Homo

sapiens

Aequorea

victoria

Escherichia

coli

Arabidopsis

thaliana

In silico gene optimization


Pagothenia

borchgrevinki

Homo

sapiens

Aequorea

victoria

Escherichia

coli

Arabidopsis

thaliana

In silico gene optimization ... is based on theuniversal code


CDS

• Codon usage• Overall GC content• Restriction sites in&out• Repetitive sequences• RNA secondary structures• mRNA halflife• Ribosome entry sites• Cryptic splice sites• Premature polyA motifs• Others ...

Computationalmulti-parameter

optimization

electronic sequence(DNA or protein)

optimized sequence

In silico gene optimization - The Gene Optimizer®


GeneOptimizer®

Wildtypesequence

Optimized

sequence

• Sequence optimization enhances performance and expression of

your genes

• Optimal codon quality for your host

• Stabilized mRNA

• Avoid unwanted motifs and secondary structures

• Reduce sequence complexity (high or low GC-content,

repetitions)

A. thaliana

E. coli

CHO

Gene optimization


3’-UTR5’-UTR

CTG

CTC

CTT

TTG

CTA

TTA

L

ATC

ATT

ATA

I

TTC

TTT

F

GAG

GAA

E ACC

ACA

ACT

ACG

T

GAG

GAA

E TGC

TGT

C

CAC

CAT

H

L

CTG

Amino Acid

Codon

Codon Quality

• Wild type not optimal for expression

• Best codon back translation not optimal w.r.t. unwanted motifs/repetitions/secondary structures

• Goal: find a tradeoff

Gene expression is influenced by many different fac tors

Considerations for sequence optimisation


Sliding window

Optimized

5’-UTR

Extended window

5’-UTR

5’-UTR

1st step

2nd step

6th step

� The sliding window moves from 5’-UTR to 3’-UTR, one codon per step

� “All possible” codon combinations (with CAI higher than a threshold) are tested

� The extended window is considered for evaluating the codon combination

� Only the first codon of the best combination is fixed

� Up to three phases with more and more relaxed thresholds

The patented sliding window algorithm


Fath et al., 2012, PLoS ONE

Mammalian expression: wildtype vs. optimized


opt > wt86 %

opt = wt

10 %

opt < wt

4 %

• All gene-optimized constructs are expressed while expression of 12 % of wildtype genes was not detectable.

• 96 % of optimized genes display equal or better expression yield thantheir wildtype counterparts.

• Up to 25-fold increase in protein expression through optimization.

average variations ≤ 10% are considered equal (opt = wt) Fath et al., 2012, PLoS ONE

Mammalian expression: wildtype vs. optimized


NiNi--purificationpurificationmockmock, , wtwt, , optopt

ElutionElutionHisHis--taggedtagged proteinsproteins

pulldownpulldownSubstrate Protein Substrate Protein

BeadsBeads

Kinase Kinase assayassay(+ATP)(+ATP)

LysisLysisTriplicateTriplicate transfectiontransfection

mockmock, , wtwt, , optopt

Western Western BlotBlot

NiNi--purificationpurificationmockmock, , wtwt, , optopt

ElutionElutionHisHis--taggedtagged proteinsproteins

pulldownpulldownSubstrate Protein Substrate Protein

BeadsBeads

Kinase Kinase assayassay(+ATP)(+ATP)

LysisLysisTriplicateTriplicate transfectiontransfection

mockmock, , wtwt, , optopt

Western Western BlotBlot

Increase of expression yields does not affect solubility or functionality

In vitro activity


ATGGCTGG....CGGTGC

Complete service chain: from gene to protein


Cloning

Oligo assembly

Oligo synthesis

Bioinformatics

Final quality control

Fragment amplification

Screening

Sequencing

DNA preparation

Size: 824 861 274 533 577 2704 2630 2680 2663 bp

0.1 - 1.0 kb 1.0 - 3.0 kb

GeneArt® Strings™: The economic version of gene synthesis


Size: 824 861 274 533 577 2704 2630 2680 2663 bp

0.1 - 1.0 kb 1.0 - 3.0 kb

GeneArt® Strings™: The economic version of gene synthesis


Strings™ 0.1 - 1.0 kb

Oligo

assembly

Oligo synthesis errors

Mutation

Enzymatic

error correction

Strings™ 1.0 - 3.0 kb

Enzymatic

error correction

... allows to get even larger

GeneArt® Strings™: Going beyond 1 kb


simple oligo assembly

with error correction

The effect of error correction on String™ synthesis

p=0.

98


GeneOptimizer® pat. pend.

wild typesequence

� 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

Summary: GeneArt® Gene Synthesis Benefits


Acknowledgements

Synthetic Biology

R&D TeamCarlsbad

Synthetic Biology

R&D TeamRegensburg

Synthetic BiologySoftware Team

Singapore

MIT - CollaborationDept Biological Engineering

Chris Voigt, Ron Weiss

38 Proprietary & Confidential The world leader in serving scienceProprietary & Confidential

Research Tools from SynBio:Genome Editing


Agenda

• Introduction• Genome and cell engineering and its applications

• Genome editing tools

• CRISPR/Cas9 based gene editing

• Multiplexed gene editing using Cas9 mRNA

• Complete workflow solutions


Genome editing with engineered nuclease

Random mutagenesis Targeted gene editing

No Precision or control Precision and Control


Random mutagenesis & integration No control on where the edits are made

RNAi-mediated Gene knock-down (posttranscriptional, hence a temporary effect on gene function)

Targeted integration, homologous or enzyme recombination Low efficiency and laborious downstream screening methods

Zinc finger technology (first-generation) Difficult to design, limitations on which genomic regions are accessible.

TAL effector technology (second-generation) Simple- to-design DNA-binding domain with one repeat per base.

Recent advances enable precision in gene engineerin gTimeline

CRISPR-Castechnology(second-generation) Small noncoding RNA–guided DNA editing

Gain precision and control

No precision or control

Programmable DNA-binding proteins/editing tools• Engineered to edit specific DNA sequence• Allow targeted gene manipulation


Genome editing with designer engineered nucleases

Trad

ition

al m

etho

dsM

oder

n/em

ergi

ng


The world leader in serving science

Rapid and efficient editing with multiplexing capabilities

CRISPR-Cas9 genome editing


CRISPR-Cas9 systemClustered regularly interspaced palindromic repeats

• What is the CRISPR system?• Prokaryotic adaptive immune response system

that confers resistance to exogenous nucleic acids (e.g., viral DNA)

• An RNA-guided DNA nuclease system

• How does it work?• Following virus invasion, bacterium integrates

small piece of foreign DNA (proto-spacer with PAM) into CRISPR loci in its chromosome

• CRISPR loci are transcribed and processed into short crRNAs that are complementary to previously encountered foreign DNA

• CRISPR RNAs form complexes with Casproteins and form the effector complexes that guide detection and cleavage of the target DNA

Figure: based on Terns and Terns (2011) Curr Opin Microbiol 14(3):321.


CRISPR-Cas9 structure - RNA-guided DNA nuclease system

PAM = NGG, NAG

Strep. pyogenes

Spacer = 18-20nt

• ds nuclease• Nickase• Binding only


How to find a PAM

12

3

54

1 TGCATTTCTCAGTCCTAAAC AGG2 GCATTTCTCAGTCCTAAACA GGG3 TCCTAAACAGGGTAATGGAC TGG4 GACTGAGAAATGCAAGACTC TGG5 CCCAGTCCATTACCCTGTTT AGG

20 nt spacer sequence PAM

Cut site

For specificity, key is to choose unique spacers within your host genome


gRNA Design

thermofisher.com/crisprdesign


(1) Oligo design for gRNA template

Precision gRNA Synthesis Kit

3’ primer

5’ primer

T7 promoter Target (20nt)

TargetR (34 nt)

xx

TargetF (34 nt)

Constant 80 nt TracrRNA5’-TAATACGACTCACTATAG NNNNNNNNNNNNNNNNNNNN GTTTTAGAGCTAGAAATAGCA…..TGAAAAAGTGGCACCGAGTCGGTGCTTTT-3’

50bp125bp

M ctrl 1 2

(3) synthetic gRNA by IVT(2) gRNA template by PCR

100bp

200bp

M ctrl 1 2

4 Hour Protocol

>10ug>200ng/ul


What has to happen for delivery?


Transfection efficiency in variety of cell lines

Cell lines Plasmid mRNA Protein

Lipid Electro Lipid Electro Lipid Electro

293FT 49 49 70 40 64 88

U2OS 15 50 21 24 18 70

Mouse ESCs 30 45 45 20 25 70

Human ESCs (H9) 0 8 20 50 0 64

Human iPSCs 0 20 66 32 0 87

N2A 66 76 66 80 66 82

Jurkat T 0 63 0 42 0 94

K562 0 45 0 27 0 72

A549 15 44 23 29 20 65

Human keratinocytes (NHEK)

0 0 0 n/a n/a 35

Human Cord blood cells CD34+

n/a n/a n/a 0 n/a 24

Observed higher cell toxicity with plasmids


Multiplex Knockout with Cas9 in Jurkat T (Male)

(A) AAVS, RelA , and HPRT targets (B) Efficiency of triple knockout

HPRT

Neg Protein

AAVS

RelA

% Cut: 0 76 79

% Cut: 0 81 88

% Cut: 0 91 90

0

0,2

0,4

0,6

0,8

1

AAVS RelA HPRT All

Zyg

osity +/+

-/-+/-

>60% knock out of 5 loci


Design to Analysis in 4 Days


Comprehensive tools & solutionsGuide to genome engineering: complete workflow

DesignIdentify target sites &

design

DeliverTransfect & analyze

DetectScreen & enrich

• GeneArt® Precision TALs

• GeneArt® CRISPR nuclease

• RNAi for gene regulation• Gene synthesis and DNA

assembly tools• Design tool utilizing state-

of-the-art algorithm built to generate optimal CRISPR & TAL designs

Gen

e E

ditin

g &

Reg

ulat

ion • Gibco® cell culture media • Transfection reagents for

DNA & RNA (Lipofectamine® 3000, Lipofectamine® 2000, Lipofectamine® MessengerMAX™)

• Cell imaging reagents and tools

Cel

l Cul

ture

&A

naly

sis

• GeneArt® Genomic Cleavage Detection Kit

• TaqMan® real-time PCR• Ion Torrent™ next-

generation sequencing• Cell sorting or bead-

based enrichmentScr

eeni

ng

Complete workflow solution• Complete set of tools and reagents for gene engineering workflow• Custom services available for each step in the workflow or the entire workflow• Cell line generation services leveraging genome editing tools and cell culture reagents


The world leader in serving science

Precise and flexible editing

TAL effector technology


TAL effector technologyTranscription activator-like (TAL) effector

• What is TAL effector technology?• Found in Xanthomonas and Ralstonia, bacterial strains that cause major crop diseases• Bacterial pathogen proteins used to rewire transcription of host plants upon infection• Up to 26 members/strain• Proteins are injected into plants via a type III secretion system• Induce expression of plant genes

Xanthomonas Leaf blight - rice Bacterial spot - tomato


TAL effector technologyHow it works

• 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

Plant cell

Bacteria

Nucleus

TranscriptsTAL effector


Why is TAL technology appealing?

• Simple code for creating engineered TALs• Customizable: Rearrangement of repeat modules allows design of

proteins with desired DNA-binding specificities to target any gene, at any position, in any genome

• They function in all cell types tested and all kingdoms of life tested


Understanding the TAL codeA simple code determines TAL DNA-binding specificity

• Each repeat consists of 34 amino acid units that are nearly identical but for 2 hypervariable residues

• 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

Repeat variable di-residue (RVD)


Understanding the TAL codeA simple code determines TAL DNA-binding specificity


Available effector domainsFor genome editing and modulation of gene expression

• Gene targeting• Gene silencing• Incorporation of exogenous DNA

Nuclease function

(Gene targeting via Fok1)

• Gene activation• Gene expressionActivator

function (Activator vp16 or vp64)

• Down-regulation gene expression (similar to the function of siRNA)

• Heritable knock-down of gene expression

Repressor function (Epigenetic

repression via KRAB)

• To target any locus in the genome with the effector domain of your choice– multiple cloning site vector

• Transient knock-down of gene expression

Custom function(Custom design via MCS

vector)

Fok1 Nuclease Pair

Activator vp16 or vp64

Repressor KRAB

MCS Vector


TAL/CRISPR-edited cell lines�Mammalian cell line services: Knock-out, Knock-in, point mutations, deletions, insertions

�We supply 5 vials containing 3x10e6 cells/vial of modified cell line

Example services

Lentivirusproduction� We can subclone into any pLenti vector

� supply 1ml concentrated lentiviralstock per construct

� HTP lentiviralproduction service also available

Sample prep�Plasmid DNA Preparation

�mRNA Synthesis

CRISPR/TAL validation�TAL design by GeneArt® Support

� We can design and subclone the target complimentary CRISPR RNA (crRNA) into the CRISPR Nuclease Vector

�In vitro Assay

�In vivo (HEK293) Assay

�Surrogate Reporter Assay


Custom services

Mammalian Stable Cell

Line Generation

cDNALibrary

Vector Modification

TOPO® Vector

Adaptation

Gateway® Vector

Adaptation

HTP Cloning

LentivirusProduction +

Concentration

Sample Prep

mRNA Synthesis

Plasmid DNA

Preparation

Custom services overview


Acknowledgements

Synthetic Biology

R&D TeamCarlsbad

Synthetic Biology

R&D TeamRegensburg

Synthetic BiologySoftware Team

Singapore

MIT - CollaborationDept Biological Engineering

Chris Voigt, Ron Weiss


© 2014 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified.

TaqMan is a registered trademark of Roche Molecular Systems, Inc. used under permission and license.



Jasons Slides


What is genome engineering?

• Genome editing is an approach whereby a genomic DNA sequence is directly changed by adding, replacing, or removing DNA bases.

Removing DNAReplacing DNAAdding DNA

• To study gene function

• To target gene mutation

• To target transgene addition for heritable modification

• To label endogenous genes

• Stable integration

• For tissue & cell engineering to produce novel functions

Gene

therapy

Disease-resistant

transgenic plants

Tissue disease

models

Animal disease

modelsStem cell

engineering

Documents

Synthetic Biology: Writing & Editing DNA€¦ · Synthetic Biology: Writing & Editing DNA. 2 Proprietary & Confidential Breeding Accelerated breeding Biotechnology ... TTA L ATC ATT