Multiplexing Genome-scale Engineering

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1 Multiplexing Genome-scale Engineering Harris Wang, Ph.D. Department of Systems Biology Department of Pathology & Cell Biology

2 Rise of Genomics

3 An Expanding Toolbox Esvelt & Wang MSB (2013)

4 Oligo-mediated Genome Editing Passy, PNAS 1999 Oligo incorporation efficiency: 25-35% per target, without selection mismatch insertion -Beta: ssdna binding protein 1,000x better than double-stranded homologous recombination Small changes, but large combinatorial libraries site-specific deletion

5 Oligo-Recombineering in Different Organisms VL6-48 Molecular Microbiology 75:138 (2010) S. cerevisiae: 3% P. aeruginosa: 1% + CRISPR DiCarlo J, Wang HH, Church G (2013) ACS Synthetic Biology Rasouly A, Johns N, Wang HH (2014) BMC Molecular Biology 10:34 (2009) Nature Methods 4(2):147 (2007) Humans: 1% Rios X, PLoS One 7:e36697 (2012) FEMS Immunol Med Microbiol 47: (2006)

6 Multiplex Automated Genome Engineering (MAGE) in silico design 1. Target multiple sites at once 2. Many variants for each site phenotype or genotype libraries of mutants 3. Continuous mutagenesis of population Wang HH, et al, Nature 460:894-8 (2009).

7 MODEST: MAGE-Oligo Design Tool Bonde M. et al, Nucleic Acid Res (2014)

8 Automated Instrumentation

9 Knowledge Design versus Evolutionary Approaches Design driven Outcome driven # of Trials

10 Engineering at the Genome-scale Transcription (promoters) Proteins (genes) GENOME Translation (RBS) Genetic code

11 Transcriptional Engineering aromatic amino acid biosynthesis Indigo Wang HH, Nature Methods 9(6):591-3 (2012)

12 Gain-of-Function Alteration of Regulatory Networks design-driven approach Wang Temme HH, K. Nature NAR (2012) Methods doi: 9(6): /nar/gks597 (2012)

13 Semi-Rational Genome Engineering targeted hypermutations

14 Translation Engineering RBS Tuning 24 genomic targets Wang HH, Nature 460:894-8 (2009).

15 Directed Evolution of a Biosynthetic Pathway Diversified for 35 cycles (phenotyping) Screen ~100k cells (genotyping) Isolation & characterization ANCESTRAL STRAINS CYCLED STRAINS EcNR2 EcHW1 EcHW2 EcHW2e EcHW2f 15 billion variants after 24 hr growth outcome-drive approach 3mm after 60 hr growth

16 Protein Engineering: MAGE-seq Kelsic E, et al, in prep 2014

17 Directed Protein Evolution infa (IF1) outcome-drive Kelsic E, et al, in prep 2014

18 MG Mb GGG GAG GCG GTG GGA E GAA GCA GTA GGC GAC GCC GTC G GGT D GAT A GCT V GTT 1692 AGG AAG ACG M ATG 2896 R AGA K AAA 9670 ACA 5967 ATA AGC AAC ACC ATC S AGT N AAT T ACT I ATT 7399 CGG CAG CCG CTG 4859 CGA Q CAA CCA 5314 CTA CGC CAC 7485 CCC CTC R CGT H CAT 9559 P CCT L CTT W TGG 321 TAG TCG TTG 1256 STOP TGA 2703 STOP TAA 9783 TCA L TTA 8816 TGC TAC TCC TTC 7048 C TGT Y TAT S TCT F TTT 321 Codon Changes w/ RF1 to free up TAG codon TAG stop TAA stop Genetic Code Engineering new trna-aa design driven

19 Genomically Recoded Organisms 321 Site-specific Mutations (TAG TAA) Efficiency of mutation Isaacs*, Carr*, Wang*, et al, Science 333: (2011).

20 Expanded AAs & Genetic Isolation of GROs new amino acids modulation of growth nnaa 1 nnaa 2 nnaa 3 wild type re.coli E.coli viruses lateral gene transfer Lajoie M, et al Science, 2013

21 Genome Engineering w/ Microarray-Oligo Libraries Bonde M, et al ACS Synth Bio, in press 2014

22 Semi-Rational Combinatorial Genome Engineering Design driven Transcription (promoters) Proteins (genes) Outcome driven GENOME Translation (RBS) Genetic code Design driven

23 Looking Forward System design and whole genome engineering methods - Combinatorial semi-rational genome engineering Measuring the impact of synthetic genetics on the host - Need for large-scale transcriptomics & metabolomics Host integration versus orthogonal systems - Need to characterize orthogonality of synthetic parts Evolutionary selection of engineered interventions - Understanding selection pressure of the cell Engineering more robust systems - Evolution resistant designs are challenging - Robust systems are often also permissive to evolution

24 Acknowledgements People Nathan Johns Victoria Stockman Sway Chen Allen Zhu (RA) Collaborators George Church (HMS), Roy Kishony (HMS), Morten Sommers (DTU)

25 THANK YOU FOR YOUR ATTENTION!