Thanks to: || || || || || || || ||

Thanks to:

.gov||||.edu||||.org||||.com|| ||

Read = = = = = = = = I/O = = = = = = = Write

1

Genome engineering: new codes, new AAs, multi-virus resistance 2-3:30pm, CLSB 521 7-Nov-2012 SB204

Azco

ArmRev.org

Oppenheimer Foundation

Gen9

LSRF

NHGRINIGMS

A revolution in reading &

writing DNA

A terabyte per bite.

4

1944 1995 2008

55

What is a minimal replicating system ?

& why should we want one?

66

Ribozyme-Catalyzed Transcription of an Active Ribozyme

Aniela Wochner, James Attwater, Alan Coulson, Philipp Holliger, Science April 2011. Evolution & engineering of an RNA polymerase ribozyme capable of synthesizing RNAs of up to 95 nt (& synthesis of a 27 nt hammerhead endonuclease ribozyme).

77

What is a minimal replicating system ?

1999 Science: Nonessential Mycoplasma genitalium protein-coding genes: 130 of 482 (+43 tRNAs)2006 PNAS: 67 confirmed. 34 added

26: disrupted only in M. pneumoniae37: mixed mutant pools in liquid culture34: Limited sampling

88

Essential genes of a minimal bacterium

PNAS 2006

99

A Whole-Cell Computational ModelPredicts Phenotype from Genotype

Karr et al. Cell 2012

1010

A Whole-Cell Computational ModelPredicts Phenotype from Genotype

Karr et al. Cell 2012

1111

Creation of a Bacterial Cell

Controlled by a Chemically

Synthesized Genome Gibson, et al 2010

Science

1 bp deletion in dnaA Not detected: Sanger 1-kb, but wrong clone sent by Blue Heron. At 10-kb level, because many of the 454 reads happened to end or start near error.

12

113 kbp DNA 151 genes

Pure translation:Forster & Church

MSB ’05 GenomeRes.’06

Shimizu, Ueda ’01

Smaller.Higher speed &

accuracy requires a few extra genes(E.coli 20 min.

doubling)

Reconstituted ribosomes:

Jewett & Church

1313

Translation

E P A

23+5S : 50S

16S : 30S

Safe Industrial Organisms Genome Engineering Rationale

1. Robust, rapid doubling 10-20 minutes2. Non-standard amino acids3. Dependence on NSAAs4. DNA non-exchangeable with environment5. Multi-virus resistance

Testing essentiality of codons (13/64)

Codon Removed Natural Role # in Genome

# in 47 / 290 Essentials # Left

TAG Stop 322 0 0

AGA/AGG Arg 4,228 5 0CTT/CTC Leu 30,030 70 1*

CCC Pro 7,401 6 0ACC Thr 31,766 151 0ATA Ile 5,797 2 0GTC Val 20,757 67 0

GCC Ala 34,747 82 0TCC Ser 11,672 88 0CGG Arg 7273 6 0TGA Stop 1,232 4 0

  Total 155,225 481 0 (Jul’12)

Green= 13 codon elimination Blue = 62 codon shuffle

Lajoie, Kosuri, Mosberg, Gregg, D.Zhang

One exception of 481 codons

ACT CTT GCC CTWG Vsr site highly specific mismatch repair

Lajoie, Kosuri, Mosberg, Gregg, D.Zhang

4 Mbp genome CAD

1919

Divide & Conquer Genome Engineering

2020

Complete Chemical Synthesis, Assembly,

and Cloning of a Mycoplasma genitalium

Genome -- Gibson, et al 2008 Science

Genome Engineering: full genome vs partial

1. E.coli > Yeast > Mycoplasma vs one species

2. Design & synthesis error consequences

3. 1E-7 genome transplantation efficiency

4. $1M vs $1 to $1k per Mb for raw DNA

22

Applications of in vitro translation

•Ribosome display•Membrane protein drug receptor studies•Personal cancer vaccines.•Labeling one protein not the whole cell.•New chemistries (e.g. mirror chirality)

Commercial Systems: Roche, Ambion, Novagen, Promega, Invitrogen, Qiagen, Stratagene, Paragon, Amersham, NEB, Sutro, EMerck

Tony Forster(Vanderbilt)

23

Mirror world :

A highly flexible tRNAacylation method fornon-natural polypeptideSynthesis. Suga lab Nature Methods 2006

Construction of Modified Ribosomes forIncorporation of d-Amino Acids into Proteins. Hecht lab Biochemistry 2006

2424

A vesicle bioreactor as a step toward an artificialcell assembly --Noireaux & Libchaber PNAS

2004

-hemolysin-eGFP in vesicleeGFP no vesicle

Genetically encoded unnatural amino acidsLiu & Schultz 2010 Ann Rev Biochem.

4. Orthogonal antibiotics(ideally inexpensive)

e.g. PEG-pAcPhe-hGH (Ambrx, Schultz) higher serum stability

Azide Strained cyclo-octyne

+

+

Prescher, JA & CR Bertozzi (2005) Nature Chem Biol. Chemistry in living systems

hydrazideKetone

triazole

hydrazone

Orthogonal AA chemistry (metabolic dependence)

e.g. PEG-pAcPhe-hGH (Ambrx, Schultz) higher serum stability

AzidePhosphine

+

+

Prescher, JA & CR Bertozzi (2005) Nature Chem Biol. Chemistry in living systems

hydroxylamineKetone

Amide

Oxime

2828

10X /year since 2005 (vs 1.5X for VLSI)

Carr & Church, Nature Biotech

Amplify pools with flanking universal primers

6 Paths to error correction1.Hyb-Select: Tian et al. 2004 Nature 2. MutS: Carr & Jacobson 2004 NAR 3. MutHLS: Smith & Modrich 1997 PNAS4. Endo/Exonuclease : Bang Nat Meth. 20085. Errase 6. Sequencing

Minimum: $500 per 1M oligos

4 Next-Gen Synthesis: on chips

8K Xeotron Photo-Generated Acid

12K Combimatrix Electrolytic

120K Roche, Febit Photolabile 5'protection

244K Agilent Ink-jet standard reagents

29

OLS bp/error SynBIOSISKosuri et al Nature Biotech

200-mer 250 130-mer 1300 130+Errase 5940

30

PCRsort&

select

pick&

place

Oligo elute amplify

Data

Oligonucleotide-Design

Roche/454

Array synthesis

Agilent OLSPolonator

multiwell plate

PicoTitrePlate

Polonatorflowcell

Rolony Photorelease

"perfect" part

2 ways to Coalesce Next-Gen DNA reading & writing

Write Read Select

micromirror

Matzas, Church, et al. (Febit,HMS) Nat. Biotech Nov 2010

31

3232

Multiplex Automated Genome Engineering

(MAGE)

Integration of genome reading & writing

Micromirror-Polonator

Wang

Terry

33

#2: ds-Linear x Circle1 step 5’>3’exo Reda/E b/T + SelectZhang et al Nat.Gen 1998 Yu et al. PNAS 2000 (GeneBridges license)

#1: ds-Circle x Circle2 step recA+ recombination

+ Select + counterselectLink et al J. Bact 1997

(Open-access)

#3: ss-90mer x ds-Circle #4: ss-Mb x ds-Circle conjugation

Costantino &Court PNAS’03 Wang et al., Nature '09

4 DNA homology-directed strategiespKO3

E.coli

MAGE

CAGE

Isaacs et al., Science ‘11

8 Optimizations for ss allele replacement

Oligo length 10x

Phosphorothioate 3x

G > –13 kcal/mol 30x

[oligo] = 0.05-50uM

Wang, et al. Nature 2009 Isaacs et al Science 2011Ellis et al PNAS 2001

Red-10,000xMutS 100x

Lagging strand: 30x

Co-selection 4x

Co-Selection: another factor of 4

Samereplichore

Locus that restores antibiotic resistance

Cross replichore

-Red ssDNA Allelic Replacement

insertion

deletion

site-specific

mismatch

37

MAGEPrimase & Nuclease

Chris

Gregg

Josh

MosbergMarc

Lajoie

5 changes per 2h4E9 genomes/day/vial

30462

22705

11512

11802

12210

9620

18664

18894

22037

16795

7016

8797

314

2765

20899

1249

15082

15272

72898

5266

9540

7490

32080

11569

17791

13399

39835

21121

28866

30530

7401

4810

41644

34568

5733

38167

12119

32265

19820

9452

24106

29581

14174

4611611924

22067

1496

2771

24991

21050

36108

14901

20813

35252

46524

27567

44217

26270

24629

5443133875

40846

15115

10774

IsaacsCharalelChurchSunWang CarrJacobsonKong Sterling

New translation code: novel AA

Safety features: no functional DNA

exchange

multi-virus resistance

39

Why genome engineering? Multi-virus resistance

Changing 13/64 codons: ACC(T), AGA(R), AGG(R), ATA(I), CCC(P), CGG(R), CTC(L), CTT(L), GCC(A), GTC(V), TAG(-), TCC(S), TGA(-)

Isaacs, Lajoie, MosbergKosuri, Wang,Carr,et al

Clinical tests of non-standard AA “orthogonal” chemistry

PEG-pAcPhe-hGH Ambrx, Cho, Schultz et al. higher serum stability

+hydrazideKetone hydrazone

4141

Improving a Natural Enzyme Activity through Incorporation ofUnnatural Amino Acids -- Ugwumba et al 2010 JACS

8-11-fold improvement … in contrast to … screening hundreds of thousands of mutants with natural amino acids.

4 Selection technologies

In vitro

GC-MSClones

Sensor-selectors

42

4 ways for in vivo coupled sensors-selectors

3. ds-DNA 4. mRNA binding

2. tRNA-ribosome1. riboswitches

43

44

68 Sensor-Selectors (old & new ligands)

Vatsan Raman

56 DNA binding proteins: ada araC arcA argPR carP cpxR crp cspA cynR cysB cytR deoR dnaA dgsA fadR farR fhlA flhCD fnr fruR fur galR gcvA glpR hipB iclR ilvY lacI lexA lrp malT marR melR metJ metR modE nagC narL narP ntrC ompR oxyR pdhR phoB purR rbsR rhaS rpoE rpoH rpoN rpoS soxS tetR torR trpR tyrR12 Riboswitches: Adenine B12 FMN Guanine Glucosamine-6-phosphate Glycine di-GMP Lysine Molybdenum PreQ1 SAM SAH TPP theophylline 3-methylxanthine

4545

.

4646

.