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Recombineering and MAGE

Nature Reviews Methods Primers volume 1, Article number: 7 (2021) Cite this article

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Abstract

Recombination-mediated genetic engineering, also known as recombineering, is the genomic incorporation of homologous single-stranded or double-stranded DNA into bacterial genomes. Recombineering and its derivative methods have radically improved genome engineering capabilities, perhaps none more so than multiplex automated genome engineering (MAGE). MAGE is representative of a set of highly multiplexed single-stranded DNA-mediated technologies. First described in Escherichia coli, both MAGE and recombineering are being rapidly translated into diverse prokaryotes and even into eukaryotic cells. Together, this modern set of tools offers the promise of radically improving the scope and throughput of experimental biology by providing powerful new methods to ease the genetic manipulation of model and non-model organisms. In this Primer, we describe recombineering and MAGE, their optimal use, their diverse applications and methods for pairing them with other genetic editing tools. We then look forward to the future of genetic engineering.

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Fig. 1: Single-stranded and double-stranded recombineering and MAGE.
Fig. 2: Optimizing the ARF in bacteria.
Fig. 3: Eukaryotic MAGE.
Fig. 4: Reading out MAGE results.
Fig. 5: Library-scale genome diversification using MAGE-seq and DIvERGE.
Fig. 6: Advanced techniques pair MAGE with other tools.
Fig. 7: Retrons allow recombineering without exogenously delivered DNA.

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Acknowledgements

The authors thank J. Aach for helpful insights. Funding for this research was provided by the US Department of Energy (DOE) under grant DE-FG02-02ER63445 (G.M.C). The authors acknowledge support from the National Institute of General Medical Sciences of the National Institutes of Health (NIH) under a Chemistry–Biology Interface Training Grant that supported M.A.J. (Award Number T32GM133395). The study was supported by the following research grants: European Research Council (ERC) H2020-ERC-2014-CoG 648364 — Resistance Evolution (C.P.); ‘Célzott Lendület’ Programme of the Hungarian Academy of Sciences LP-2017–10/2017 (C.P.); ‘Élvonal’ KKP 126506 (C.P.); and GINOP-2.3.2–15–2016–00014 (EVOMER, to C.P.). P.N.C. was supported by Physical and Engineering Biology training grant 5T32EB019941-05. A.N. was supported by an EMBO LTF 160-2019 Long-Term fellowship. A.D.E. and K.J. acknowledge funding from the Air Force Office of Scientific Research (FA9550-14-1-0089) and the Welch Foundation (F-1654).

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Authors and Affiliations

  1. Department of Genetics, Harvard Medical School, Boston, MA, USA

    Timothy M. Wannier, Akos Nyerges, Max G. Schubert & George M. Church

  2. Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, MA, USA

    Timothy M. Wannier, Gabriel T. Filsinger, Akos Nyerges, Max G. Schubert & George M. Church

  3. Department of Molecular, Cellular & Developmental Biology, Yale University, New Haven, CT, USA

    Peter N. Ciaccia & Farren J. Isaacs

  4. Systems Biology Institute, Yale University, West Haven, CT, USA

    Peter N. Ciaccia & Farren J. Isaacs

  5. Department of Molecular Biosciences, College of Natural Sciences, University of Texas at Austin, Austin, TX, USA

    Andrew D. Ellington & Kamyab Javanmardi

  6. Department of Systems Biology, Harvard University, Cambridge, MA, USA

    Gabriel T. Filsinger

  7. Department of Biomedical Engineering, Yale University, New Haven, CT, USA

    Farren J. Isaacs

  8. Department of Chemical and Biomolecular Engineering, University of Delaware, Newark, DE, USA

    Michaela A. Jones & Aditya M. Kunjapur

  9. Synthetic and Systems Biology Unit, Institute of Biochemistry, Biological Research Centre, Szeged, Hungary

    Csaba Pal

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  1. Timothy M. Wannier
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Contributions

Introduction (T.M.W., P.N.C. and F.J.I.); Experimentation (T.M.W., P.N.C., F.J.I. and C.P.); Results (T.M.W.); Applications (T.M.W., A.D.E., K.J., M.A.J., A.M.K., A.N. and M.G.S.); Reproducibility and data deposition (T.M.W.); Limitations and optimizations (T.M.W. and A.N.); Outlook (T.M.W., G.T.F. and G.M.C.). Overview of the Primer (T.M.W. and G.T.F.).

Corresponding author

Correspondence to Timothy M. Wannier.

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Competing interests

T.M.W, G.T.F. and G.M.C. are inventors on a patent application related to serial enrichment for efficient recombineering (SEER) and new single-stranded DNA-annealing protein (SSAP) discovery. A.N. and C.P. are inventors on a patent related to directed evolution with random genomic mutations (DIvERGE) (US10669537B2: Mutagenizing Intracellular Nucleic Acids). F.J.I. and G.M.C. are inventors on a MAGE patent, which has been licensed. F.J.I. is an inventor on a patent application related to eukaryotic MAGE. The remaining authors declare no competing interests.

Additional information

Peer review information

Nature Reviews Methods Primers thanks N. Claassens, A. Garst, M. Lluch Senar, J. Yang and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Related links

European Nucleotide Archive (ENA): https://www.ebi.ac.uk/ena/browser

GitHub: https://github.com

National Center for Biotechnology Information (NCBI) Sequence Read Archive (SRA): https://www.ncbi.nlm.nih.gov/sra

Glossary

Homologous recombination

A type of genetic recombination by which nucleotide sequences are exchanged between molecules that share similar or identical sequences.

DNA double-strand breaks

(DSBs). Simultaneous breaks in both strands of a DNA helix.

Non-homologous end-joining

(NHEJ). The repair of double-strand DNA breaks by direct ligation of cut DNA ends without a homologous template.

Short guide RNAs

(gRNAs). Molecules that bind to and then guide Cas9 or a similar protein to a targeted genomic locus by nucleotide base pairing.

Homology-directed repair

(HDR). The repair of double-strand DNA breaks using a homologous template.

Combinatorial genetic diversity

A population of cells that has been diversified through genetic engineering to include individual cells that each contain multiple modifications to their genome. These modifications are randomly introduced from a pool of potential modifications, creating combinatorial diversity in the population.

Base editing

A method that fuses a Cas9 nickase to a deaminase domain. The Cas9 is directed by a guide RNA to a target site on the genome, whereupon the deaminase will edit within a window of DNA bases.

Prime editing

A method whereby a Cas9 nickase is fused to a reverse transcriptase and a guide RNA is fused to a repair template. The Cas9 nickase nicks the target DNA strand, is then resected by host proteins and the reverse-transcribed DNA is used as a repair template, conveying the specified modification.

Cas9 nickase

A Cas9 variant that has been partially deactivated so that it cuts one strand of a double-stranded helix, creating a ‘nick’ instead of a double-strand break.

Reverse transcriptase

An enzyme that transcribes RNA into cDNA.

Single-nucleotide polymorphisms

Any number of substitutions of single nucleotides at specific genomic locations.

Reverse genetics

Classical genetics is the prediction of allelic determinants of phenotypic variation by genetic analysis. Reverse genetics is the creation of genetic variation and subsequent phenotypic characterization of these known allelic variants.

Single-stranded DNA-annealing protein

(SSAP). A protein that speeds the specific annealing of two strands of single-stranded DNA (ssDNA), sometimes also interacting with proteins coating ssDNA to allow annealing to proceed.

Multiplex automated genome engineering

(MAGE). An umbrella term referring to techniques that involve single-stranded DNA-mediated recombineering at multiple sites.

ssDNA recombineering

Recombineering using single-stranded DNA (ssDNA) as the carrier of genetic information.

Whole-genome recoding

The replacement of a codon with one or multiple alternative codons systematically throughout a genome.

Bacterial artificial chromosome

A large circular DNA element distinct from the bacterial chromosome that replicates from a plasmid origin.

Allelic recombination frequency

(ARF). The fraction of a cell population that successfully inherits a specified modification after a genetic editing technique such as multiplex automated genome engineering is carried out.

Single-stranded DNA-binding protein

(SSB). An essential protein that binds to single-stranded DNA, protecting it and coordinating chromosome replication, and that is preserved throughout all domains of life.

Serial enrichment for efficient recombineering

(SEER). A method for screening a large library of single-stranded DNA-annealing proteins to identify variants that perform efficiently in a given host.

Single-stranded DNA annealing

The annealing of two strands of single-stranded DNA by base pairing.

Co-selection MAGE

A multiplex genome engineering technique in which a target modification that does not confer a selective phenotype is made in close proximity to one that does, allowing enrichment of both modifications in comparison with an unselected population.

Origin of replication

The site at which proteins involved in genome replication begin the synthesis of a new genomic copy.

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Wannier, T.M., Ciaccia, P.N., Ellington, A.D. et al. Recombineering and MAGE. Nat Rev Methods Primers 1, 7 (2021). https://doi.org/10.1038/s43586-020-00006-x

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