1. 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 NHGRI NIGMS

2. A revolution in reading & writing DNA

3. A terabyte per bite.

4. 4 1944 1995 2008

5. 55 What is a minimal replicating system ? & why should we want one?

6. 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).

7. 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. pneumoniae 37: mixed mutant pools in liquid culture 34: Limited sampling

8. 88 Essential genes of a minimal bacterium PNAS 2006

9. 99 A Whole-Cell Computational Model Predicts Phenotype from Genotype Karr et al. Cell 2012

10. 10 A Whole-Cell Computational Model Predicts Phenotype from Genotype Karr et al. Cell 2012

11. 11 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. 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

13. 13 Translation E P A 23+5S : 50S 16S : 30S

14. Safe Industrial Organisms Genome Engineering Rationale 1.Robust, rapid doubling 10-20 minutes 2.Non-standard amino acids 3.Dependence on NSAAs 4.DNA non-exchangeable with environment 5.Multi-virus resistance

15. Testing essentiality of codons (13/64) Codon RemovedNatural Role# in Genome # in 47 / 290 Essentials# Left TAGStop32200 AGA/AGGArg4,22850 CTT/CTCLeu30,030701* CCCPro7,40160 ACCThr31,7661510 ATAIle5,79720 GTCVal20,757670 GCCAla34,747820 TCCSer11,672880 CGGArg727360 TGAStop1,23240 Total155,2254810 (Jul’12)

16. Green= 13 codon elimination Blue = 62 codon shuffle  Lajoie, Kosuri, Mosberg, Gregg, D.Zhang

17. One exception of 481 codons ACT CTT GCC CTWG Vsr site highly specific mismatch repair Lajoie, Kosuri, Mosberg, Gregg, D.Zhang

18. 4 Mbp genome CAD

19. 19 Divide & Conquer Genome Engineering

20. 20 Complete Chemical Synthesis, Assembly, and Cloning of a Mycoplasma genitalium Genome -- Gibson, et al 2008 Science

21. 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. 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. 23 Mirror world : A highly flexible tRNA acylation method for non-natural polypeptide Synthesis. Suga lab Nature Methods 2006 Construction of Modified Ribosomes for Incorporation of d-Amino Acids into Proteins. Hecht lab Biochemistry 2006

24. 24 A vesicle bioreactor as a step toward an artificial cell assembly --Noireaux & Libchaber PNAS 2004  -hemolysin-eGFP in vesicle eGFP no vesicle

25. Genetically encoded unnatural amino acids Liu & Schultz 2010 Ann Rev Biochem.

26. 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 hydrazide Ketone triazole hydrazone

27. Orthogonal AA chemistry (metabolic dependence) e.g. PEG-pAcPhe-hGH (Ambrx, Schultz) higher serum stability Azide Phosphine + + Prescher, JA & CR Bertozzi (2005) Nature Chem Biol. Chemistry in living systems hydroxylamine Ketone Amide Oxime

28. 28 10X /year since 2005 (vs 1.5X for VLSI) Carr & Church, Nature Biotech

29. Amplify pools with flanking universal primers 6 Paths to error correction 1.Hyb-Select: Tian et al. 2004 Nature 2. MutS: Carr & Jacobson 2004 NAR 3. MutHLS: Smith & Modrich 1997 PNAS 4. Endo/Exonuclease : Bang Nat Meth. 2008 5. 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

30. OLS bp/error SynBIOSIS Kosuri et al Nature Biotech 200-mer 250 130-mer 1300 130+Errase 5940 30

31. PCR sort & select pick & place Oligo elute amplify Data Oligonucleotide-Design Roche/454 Array synthesis Agilent OLS Polonator multiwell plate PicoTitrePlate Polonator flowcell Rolony Photorelease "perfect" part 2 ways to Coalesce Next-Gen DNA reading & writing WriteReadSelect micromirror Matzas, Church, et al. (Febit,HMS) Nat. Biotech Nov 2010 31

32. 32 Multiplex Automated Genome Engineering (MAGE) Integration of genome reading & writing Micromirror- Polonator Wang Terry

33. 33 #2: ds-Linear x Circle 1 step 5’>3’exo Reda/E b/T + Select Zhang et al Nat.Gen 1998 Yu et al. PNAS 2000 (GeneBridges license) #1: ds-Circle x Circle 2 step recA+ recombination + Select + counterselect Link 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 strategies pKO3 E.coli MAGE CAGE Isaacs et al., Science ‘11

34. 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 2011 Ellis et al PNAS 2001 Red-  10,000x MutS 100x Lagging strand: 30x Co-selection 4x

35. Co-Selection: another factor of 4 Same replichore Locus that restores antibiotic resistance Cross replichore

36. -Red ssDNA Allelic Replacement insertion deletion site-specific mismatch

37. 37 MAGE Primase & Nuclease Chris Gregg Josh Mosberg Marc Lajoie 5 changes per 2h 4E9 genomes/day/vial

38. 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 46116 11924 22067 1496 2771 24991 21050 36108 14901 20813 35252 46524 27567 44217 26270 24629 54431 33875 40846 15115 10774 Isaacs Charalel Church Sun Wang Carr Jacobson Kong Sterling New translation code: novel AA Safety features: no functional DNA exchange multi-virus resistance

39. 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, Mosberg Kosuri, Wang, Carr, et al

40. Clinical tests of non-standard AA “orthogonal” chemistry PEG-pAcPhe-hGH Ambrx, Cho, Schultz et al. higher serum stability + hydrazide Ketone hydrazone

41. 41 Improving a Natural Enzyme Activity through Incorporation of Unnatural Amino Acids -- Ugwumba et al 2010 JACS 8-11-fold improvement … in contrast to … screening hundreds of thousands of mutants with natural amino acids.

42. 4 Selection technologies In vitro GC-MS Clones Sensor- selectors 42

43. 4 ways for in vivo coupled sensors-selectors 3. ds-DNA 4. mRNA binding 2. tRNA-ribosome 1. riboswitches 43

44. 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 tyrR 12 Riboswitches: Adenine B12 FMN Guanine Glucosamine-6- phosphate Glycine di-GMP Lysine Molybdenum PreQ1 SAM SAH TPP theophylline 3-methylxanthine

45. 45.

46. 46.