Genome engineering new codes new AAs multi-virus resistance

Genome engineering: new codes, new AAs, multi-virus resistance 23: 30 pm, CLSB 521 7 -Nov-2012 SB 204 Thanks to: . gov NHGRI || NIGMS ||. edu || Arm. Rev. org || Oppenheimer . org Foundation || ||. com Azco || || Read LSRF Gen 9 = = = = I/O = = = = Write 1

A revolution in reading & writing DNA

A terabyte per bite.

1944 1995 2008 4

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

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). 6

What is a minimal replicating system ? 1999 Science: Nonessential Mycoplasma genitalium proteincoding genes: 130 of 482 (+43 t. RNAs) 2006 PNAS: 67 confirmed. 34 added 26: disrupted only in M. pneumoniae 37: mixed mutant pools in liquid culture 34: Limited sampling 7

Essential genes of a minimal bacterium PNAS 2006 8

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

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

Creation of a Bacterial Cell Controlled by a Chemically Synthesized Genome Gibson, et al 2010 Science 1 bp deletion in dna. A 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. 11

113 kbp DNA 151 genes Smaller. Higher speed & accuracy requires a few extra genes (E. coli 20 min. doubling) Reconstituted ribosomes: Jewett & Church Pure translation: Forster & Church MSB ’ 05 Genome. Res. ’ 06 Shimizu, Ueda ’ 01 12

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

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

Testing essentiality of codons (13/64) Codon Removed TAG # in 47 / 290 Natural Role # in Genome Essentials Stop 322 0 # Left 0 AGA/AGG CTT/CTC CCC ATA GTC GCC TCC CGG TGA Arg Leu Pro Thr Ile Val Ala Ser Arg Stop 4, 228 30, 030 7, 401 31, 766 5, 797 20, 757 34, 747 11, 672 7273 1, 232 5 70 6 151 2 67 82 88 6 4 0 1* 0 0 0 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

Divide & Conquer Genome Engineering 19

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

Genome Engineering: full genome vs partial 1. E. coli > Yeast > Mycoplasma vs one species 2. Design & synthesis error consequences 3. 1 E-7 genome transplantation efficiency 4. $1 M vs $1 to $1 k per Mb for raw DNA

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) 22

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

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

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

4. Orthogonal antibiotics (ideally inexpensive) Azide Strained cyclo-octyne triazole + Ketone + hydrazide hydrazone e. g. PEG-p. Ac. Phe-h. GH (Ambrx, Schultz) higher serum stability Prescher, JA & CR Bertozzi (2005) Nature Chem Biol. Chemistry in living systems

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

10 X /year since 2005 (vs 1. 5 X for VLSI) Carr & Church, Nature Biotech 28

4 Next-Gen Synthesis: on chips Minimum: $500 per 1 M oligos 8 K Xeotron Photo-Generated Acid 12 K Combimatrix Electrolytic 120 K Roche, Febit Photolabile 5'protection 244 K Agilent Ink-jet standard reagents Amplify pools with flanking universal primers 6 Paths to error correction 1. Hyb-Select: Tian et al. 2004 Nature 2. Mut. S: Carr & Jacobson 2004 NAR 3. Mut. HLS: Smith & Modrich 1997 PNAS 4. Endo/Exonuclease : Bang Nat Meth. 2008 5. Errase 6. Sequencing 29

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

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

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

4 DNA homology-directed strategies p. KO 3 E. coli #1: ds-Circle x Circle 2 step rec. A+ recombination + Select + counterselect Link et al J. Bact 1997 (Open-access) #3: ss-90 mer x ds-Circle #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 (Gene. Bridges license) #4: ss-Mb x ds-Circle conjugation Costantino &Court PNAS’ 03 Isaacs et al. , Science ‘ 11 Wang et al. , Nature '09 CAGE MAGE 33

8 Optimizations for ss allele replacement Mut. S 100 x Oligo length 10 x Red-b >10, 000 x Co-selection 4 x Lagging strand: 30 x G > – 13 kcal/mol 30 x Phosphorothioate 3 x [oligo] = 0. 05 -50 u. M Wang, et al. Nature 2009 Isaacs et al Science 2011 Ellis et al PNAS 2001

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

-Red ss. DNA Allelic Replacement mismatch insertion site-specific deletion

MAGE Primase & Nuclease 5 changes per 2 h 4 E 9 genomes/day/vial Chris Josh Gregg Mosberg Marc Lajoie 37

22705 11802 16795 8797 30462 11512 22037 7016 18894 9620 18664 12210 1249 2765 314 New translation code: novel AA Safety features: no functional DNA exchange multi-virus resistance 20899 15272 7490 13399 30530 15082 9540 17791 28866 4810 5266 11569 21121 72898 32080 39835 29581 34568 32265 41644 12119 7401 22067 9452 38167 11924 2771 19820 5733 24106 46116 14174 1496 21050 35252 26270 40846 24991 20813 33875 10774 15115 14901 27567 44217 54431 36108 46524 24629 Isaacs Charalel Church Sun Wang Carr Jacobson Kong Sterling

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 39

Clinical tests of non-standard AA “orthogonal” chemistry hydrazide Ketone hydrazone + PEG-p. Ac. Phe-h. GH Ambrx, Cho, Schultz et al. higher serum stability

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. 41

4 Selection technologies In vitro Clones GC-MS Sensorselectors 42

4 ways for in vivo coupled sensors-selectors 1. riboswitches 3. ds-DNA 2. t. RNA-ribosome 4. m. RNA binding 43

68 Sensor-Selectors (old & new ligands) 56 DNA binding proteins: ada ara. C arc. A arg. PR car. P cpx. R crp csp. A cyn. R cys. B cyt. R deo. R dna. A dgs. A fad. R far. R fhl. A flh. CD fnr fru. R fur gal. R gcv. A glp. R hip. B icl. R ilv. Y lac. I lex. A lrp mal. T mar. R mel. R met. J met. R mod. E nag. C nar. L nar. P ntr. C omp. R oxy. R pdh. R pho. B pur. R rbs. R rha. S rpo. E rpo. H rpo. N rpo. S sox. S tet. R tor. R trp. R tyr. R 12 Riboswitches: Adenine B 12 FMN Guanine Glucosamine-6 phosphate Glycine di-GMP Lysine Molybdenum Pre. Q 1 SAM SAH TPP theophylline 3 -methylxanthine Vatsan Raman 44

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