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Strategies for cloning and manipulating natural and synthetic chromosomes

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Abstract

Advances in synthetic biology methods to assemble and edit DNA are enabling genome engineering at a previously impracticable scale and scope. The synthesis of the Mycoplasma mycoides genome followed by its transplantation to convert a related cell into M. mycoides has transformed strain engineering. This approach exemplifies the combination of newly emerging chromosome-scale genome editing strategies that can be defined in three main steps: (1) chromosome acquisition into a microbial engineering platform, (2) alteration and improvement of the acquired chromosome, and (3) installation of the modified chromosome into the original or alternative organism. In this review, we outline recent progress in methods for acquiring chromosomes and chromosome-scale DNA molecules in the workhorse organisms Bacillus subtilis, Escherichia coli, and Saccharomyces cerevisiae. We present overviews of important genetic strategies and tools for each of the three organisms, point out their respective strengths and weaknesses, and highlight how the host systems can be used in combination to facilitate chromosome assembly or engineering. Finally, we highlight efforts for the installation of the cloned/altered chromosomes or fragments into the target organism and present remaining challenges in expanding this powerful experimental approach to a wider range of target organisms.

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Abbreviations

AAI:

Acquire, alter, and install

YAC:

Yeast artificial chromosome

Bsu168:

B. subtilis strain Marburg 168

BGM:

Bacillus GenoMe

IWe:

Inchworm elongation

OGAB:

Ordered gene assembly in Bsu168

BACs:

Bacterial artificial chromosomes

PCR:

Polymerase chain reaction

MAGE:

Multiplex automated genome engineering

CAGE:

Conjugative assembly genome engineering

ARS:

Autonomously replicating sequence

ORC:

Origin recognition complex

TAR:

Transformation-associated recombination

5-FOA:

5-Fluoroorotic acid

TREC:

Tandem repeat coupled with endonuclease cleavage

DSB:

Double-stranded break

TALENs:

Transcription activator-like effector nucleases

CRISPRs:

Clustered regularly interspersed short palindromic repeats

TALE:

Transcription activator-like effector

gRNA:

Guide RNA

PEG:

Polyethylene glycol

References

  • Annaluru N, Muller H, Mitchell LA et al (2014) Total synthesis of a functional designer eukaryotic chromosome. Science 344:55–58

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Barnett MJ, Fisher RF, Jones T et al (2001) Nucleotide sequence and predicted functions of the entire Sinorhizobium meliloti pSymA megaplasmid. Proc Natl Acad Sci U S A 98:9883–9888

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Benders GA, Noskov VN, Denisova EA et al (2010) Cloning whole bacterial genomes in yeast. Nucleic Acids Res 38:2558–2569

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Boeke JD, Lacroute F, Fink GR (1984) A positive selection for mutants lacking orotidine-5′-phosphate decarboxylase activity in yeast: 5-fluoro-orotic acid resistance. Mol Gen Genet 197:345–346

    Article  CAS  PubMed  Google Scholar 

  • Chan KM, Liu YT, Ma CH, Jayaram M, Sau S (2013) The 2 micron plasmid of Saccharomyces cerevisiae: a miniaturized selfish genome with optimized functional competence. Plasmid 70:2–17

    Article  CAS  PubMed  Google Scholar 

  • Cong L, Ran FA, Cox D et al (2013) Multiplex genome engineering using CRISPR/Cas systems. Science 339:819–823

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Datsenko KA, Wanner BL (2000) One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc Natl Acad Sci U S A 97:6640–6645

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Dicarlo JE, Conley AJ, Penttila M et al (2013) Yeast oligo-mediated genome engineering (YOGE). ACS Synth Biol 2:741–749

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Dymond JS, Richardson SM, Coombes CE et al (2011) Synthetic chromosome arms function in yeast and generate phenotypic diversity by design. Nature 477:471–476

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Esvelt KM, Wang HH (2013) Genome-scale engineering for systems and synthetic biology. Mol Syst Biol 9:641

    Article  PubMed Central  PubMed  Google Scholar 

  • Gibson DG (2014) Programming biological operating systems: genome design, assembly and activation. Nat Methods 11:521–526

    Article  CAS  PubMed  Google Scholar 

  • Gibson DG, Benders GA, Andrews-Pfannkoch C et al (2008a) Complete chemical synthesis, assembly, and cloning of a Mycoplasma genitalium genome. Science 319:1215–1220

    Article  CAS  PubMed  Google Scholar 

  • Gibson DG, Benders GA, Axelrod KC et al (2008b) One-step assembly in yeast of 25 overlapping DNA fragments to form a complete synthetic Mycoplasma genitalium genome. Proc Natl Acad Sci U S A 105:20404–20409

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Gibson DG, Glass JI, Lartigue C et al (2010a) Creation of a bacterial cell controlled by a chemically synthesized genome. Science 329:52–56

    Article  CAS  PubMed  Google Scholar 

  • Gibson DG, Smith HO, Hutchison CA 3rd, Venter JC, Merryman C (2010b) Chemical synthesis of the mouse mitochondrial genome. Nat Methods 7:901–903

    Article  CAS  PubMed  Google Scholar 

  • Gietz RD (2014) Yeast transformation by the LiAc/SS carrier DNA/PEG method. Methods Mol Biol 1163:33–44

    Article  PubMed  Google Scholar 

  • Gyuris J, Duda EG (1986) High-efficiency transformation of Saccharomyces cerevisiae cells by bacterial minicell protoplast fusion. Mol Cell Biol 6:3295–3297

    PubMed Central  CAS  PubMed  Google Scholar 

  • Hinnen A, Hicks JB, Fink GR (1978) Transformation of yeast. Proc Natl Acad Sci U S A 75:1929–1933

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Holt RA, Warren R, Flibotte S et al (2007) Rebuilding microbial genomes. Bioessays 29:580–590

  • Hsu PD, Lander ES, Zhang F (2014) Development and applications of CRISPR-Cas9 for genome engineering. Cell 157:1262–1278

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Isaacs FJ, Carr PA, Wang HH et al (2011) Precise manipulation of chromosomes in vivo enables genome-wide codon replacement. Science 333:348–353

    Article  CAS  PubMed  Google Scholar 

  • Itaya M, Nagata T, Shiroishi T, Fujita K, Tsuge K (2000) Efficient cloning and engineering of giant DNAs in a novel Bacillus subtilis genome vector. J Biochem 128:869–875

    Article  CAS  PubMed  Google Scholar 

  • Itaya M, Fujita K, Ikeuchi M, Koizumi M, Tsuge K (2003) Stable positional cloning of long continuous DNA in the Bacillus subtilis genome vector. J Biochem 134:513–519

    Article  CAS  PubMed  Google Scholar 

  • Itaya M, Tsuge K, Koizumi M, Fujita K (2005) Combining two genomes in one cell: stable cloning of the Synechocystis PCC6803 genome in the Bacillus subtilis 168 genome. Proc Natl Acad Sci U S A 102:15971–15976

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Itaya M, Fujita K, Kuroki A, Tsuge K (2008) Bottom-up genome assembly using the Bacillus subtilis genome vector. Nat Methods 5:41–43

    Article  CAS  PubMed  Google Scholar 

  • Johnston C, Martin B, Fichant G, Polard P, Claverys JP (2014) Bacterial transformation: distribution, shared mechanisms and divergent control. Nat Rev Microbiol 12:181–196

    Article  CAS  PubMed  Google Scholar 

  • Kaneko S, Itaya M (2010) Designed horizontal transfer of stable giant DNA released from Escherichia coli. J Biochem 147:819–822

    Article  CAS  PubMed  Google Scholar 

  • Karas BJ, Tagwerker C, Yonemoto IT, Hutchison CA 3rd, Smith HO (2012) Cloning the Acholeplasma laidlawii PG-8A genome in Saccharomyces cerevisiae as a yeast centromeric plasmid. ACS Synth Biol 1:22–28

    Article  CAS  PubMed  Google Scholar 

  • Karas BJ, Jablanovic J, Sun L et al (2013a) Direct transfer of whole genomes from bacteria to yeast. Nat Methods 10:410–412

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Karas BJ, Molparia B, Jablanovic J et al (2013b) Assembly of eukaryotic algal chromosomes in yeast. J Biol Eng 7:30

    Article  PubMed Central  PubMed  Google Scholar 

  • Karas BJ, Jablanovic J, Irvine E et al (2014a) Transferring whole genomes from bacteria to yeast spheroplasts using entire bacterial cells to reduce DNA shearing. Nat Protoc 9:743–750

    Article  CAS  PubMed  Google Scholar 

  • Karas BJ, Wise KS, Sun L et al (2014b) Rescue of mutant fitness defects using in vitro reconstituted designer transposons in Mycoplasma mycoides. Front Microbiol 5:369

    Article  PubMed Central  PubMed  Google Scholar 

  • Kim H, Kim JS (2014) A guide to genome engineering with programmable nucleases. Nat Rev Genet 15:321–334

    Article  CAS  PubMed  Google Scholar 

  • Kouprina N, Larionov V (2006) TAR cloning: insights into gene function, long-range haplotypes and genome structure and evolution. Nat Rev Genet 7:805–812

    Article  CAS  PubMed  Google Scholar 

  • Kouprina N, Larionov V (2008) Selective isolation of genomic loci from complex genomes by transformation-associated recombination cloning in the yeast Saccharomyces cerevisiae. Nat Protoc 3:371–377

    Article  CAS  PubMed  Google Scholar 

  • Krishnakumar R, Grose C, Haft DH et al (2014) Simultaneous non-contiguous deletions using large synthetic DNA and site-specific recombinases. Nucleic Acids Res 42:e111

    Article  PubMed Central  PubMed  Google Scholar 

  • Lajoie MJ, Gregg CJ, Mosberg JA, Washington GC, Church GM (2012) Manipulating replisome dynamics to enhance lambda Red-mediated multiplex genome engineering. Nucleic Acids Res 40:e170

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Lartigue C, Glass JI, Alperovich N et al (2007) Genome transplantation in bacteria: changing one species to another. Science 317:632–638

    Article  CAS  PubMed  Google Scholar 

  • Lartigue C, Vashee S, Algire MA et al (2009) Creating bacterial strains from genomes that have been cloned and engineered in yeast. Science 325:1693–1696

    Article  CAS  PubMed  Google Scholar 

  • Lee BR, Cho S, Song Y, Kim SC, Cho BK (2013) Emerging tools for synthetic genome design. Mol Cells 35:359–370

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Mosberg JA, Gregg CJ, Lajoie MJ, Wang HH, Church GM (2012) Improving lambda red genome engineering in Escherichia coli via rational removal of endogenous nucleases. PLoS One 7:e44638

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Newlon CS, Theis JF (1993) The structure and function of yeast ARS elements. Curr Opin Genet Dev 3:752–758

    Article  CAS  PubMed  Google Scholar 

  • Nishizaki T, Tsuge K, Itaya M, Doi N, Yanagawa H (2007) Metabolic engineering of carotenoid biosynthesis in Escherichia coli by ordered gene assembly in Bacillus subtilis. Appl Environ Microbiol 73:1355–1361

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Noskov VN, Segall-Shapiro TH, Chuang RY (2010) Tandem repeat coupled with endonuclease cleavage (TREC): a seamless modification tool for genome engineering in yeast. Nucleic Acids Res 38:2570–2576

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Noskov VN, Karas BJ, Young L et al (2012) Assembly of large, high G + C bacterial DNA fragments in yeast. ACS Synth Biol 1:267–273

    Article  CAS  PubMed  Google Scholar 

  • O’Neill BM, Mikkelson KL, Gutierrez NM et al (2012) An exogenous chloroplast genome for complex sequence manipulation in algae. Nucleic Acids Res 40:2782–2792

    Article  PubMed Central  PubMed  Google Scholar 

  • Pinel D, D’Aoust F, Del Cardayre SB et al (2011) Saccharomyces cerevisiae genome shuffling through recursive population mating leads to improved tolerance to spent sulfite liquor. Appl Environ Microbiol 77:4736–4743

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Ran FA, Hsu PD, Lin CY et al (2013) Double nicking by RNA-guided CRISPR Cas9 for enhanced genome editing specificity. Cell 154:1380–1389

    Article  CAS  PubMed  Google Scholar 

  • Sanjana NE, Cong L, Zhou Y et al (2012) A transcription activator-like effector toolbox for genome engineering. Nat Protoc 7:171–192

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Sorek R, Zhu Y, Creevey CJ et al (2007) Genome-wide experimental determination of barriers to horizontal gene transfer. Science 318:1449–1452

  • Storici F, Lewis LK, Resnick MA (2001) In vivo site-directed mutagenesis using oligonucleotides. Nat Biotechnol 19:773–776

    Article  CAS  PubMed  Google Scholar 

  • Suzuki Y, St Onge RP, Mani R et al (2011) Knocking out multigene redundancies via cycles of sexual assortment and fluorescence selection. Nat Methods 8:159–164

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Tagwerker C, Dupont CL, Karas BJ et al (2012) Sequence analysis of a complete 1.66 Mb Prochlorococcus marinus MED4 genome cloned in yeast. Nucleic Acids Res 40:10375–10383

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Tischer BK, Von Einem J, Kaufer B, Osterrieder N (2006) Two-step red-mediated recombination for versatile high-efficiency markerless DNA manipulation in Escherichia coli. Biotechniques 40:191–197

    Article  CAS  PubMed  Google Scholar 

  • Tsuge K, Matsui K, Itaya M (2003) One step assembly of multiple DNA fragments with a designed order and orientation in Bacillus subtilis plasmid. Nucleic Acids Res 31:e133

    Article  PubMed Central  PubMed  Google Scholar 

  • Tsuge K, Matsui K, Itaya M (2007) Production of the non-ribosomal peptide plipastatin in Bacillus subtilis regulated by three relevant gene blocks assembled in a single movable DNA segment. J Biotechnol 129:592–603

    Article  CAS  PubMed  Google Scholar 

  • Wang HH, Isaacs FJ, Carr PA et al (2009) Programming cells by multiplex genome engineering and accelerated evolution. Nature 460:894–898

    Article  CAS  PubMed  Google Scholar 

  • Warren RL, Freeman JD, Levesque RC et al (2008) Transcription of foreign DNA in Escherichia coli. Genome Res 18:1798–1805

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Yamanaka K, Reynolds KA, Kersten RD et al (2014) Direct cloning and refactoring of a silent lipopeptide biosynthetic gene cluster yields the antibiotic taromycin A. Proc Natl Acad Sci U S A 111:1957–1962

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Yang XW, Model P, Heintz N (1997) Homologous recombination based modification in Escherichia coli and germline transmission in transgenic mice of a bacterial artificial chromosome. Nat Biotechnol 15:859–865

    Article  CAS  PubMed  Google Scholar 

  • Yu D, Ellis HM, Lee EC et al (2000) An efficient recombination system for chromosome engineering in Escherichia coli. Proc Natl Acad Sci U S A 97:5978–5983

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Yu BJ, Kang KH, Lee JH et al (2008) Rapid and efficient construction of markerless deletions in the Escherichia coli genome. Nucleic Acids Res 36:e84

    Article  PubMed Central  PubMed  Google Scholar 

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Acknowledgments

This work was supported by the United States Department of Energy Genomics Science program grant (DE-SC0008593) (P. D. W. and B. J. K.) and by the United States Department of Energy cooperative agreement DE-EE0006109 (Y. S.).

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Authors and Affiliations

  1. Synthetic Biology and Bioenergy Group, J. Craig Venter Institute, 4120 Capricorn Lane, La Jolla, CA, 92037, USA

    Bogumil J. Karas, Yo Suzuki & Philip D. Weyman

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  1. Bogumil J. Karas
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  2. Yo Suzuki
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  3. Philip D. Weyman
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Correspondence to Philip D. Weyman.

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Responsible Editors: Natalay Kouprina and Vladimir Larionov.

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Karas, B.J., Suzuki, Y. & Weyman, P.D. Strategies for cloning and manipulating natural and synthetic chromosomes. Chromosome Res 23, 57–68 (2015). https://doi.org/10.1007/s10577-014-9455-3

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