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High throughput functional variant screens via in-vivo production of single-stranded DNA

View ORCID ProfileMax G. Schubert, Daniel B. Goodman, Timothy M. Wannier, Divjot Kaur, Fahim Farzadfard, Timothy K. Lu, Seth L. Shipman, George M. Church
doi: https://doi.org/10.1101/2020.03.05.975441
Max G. Schubert
1Harvard University, Boston, MA, USA
6Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, MA
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  • ORCID record for Max G. Schubert
  • For correspondence: mgschubert@gmail.com
Daniel B. Goodman
2University of California, San Francisco, San Francisco, CA, USA
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Timothy M. Wannier
1Harvard University, Boston, MA, USA
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Divjot Kaur
3Universtity of Warwick, United Kingdom
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Fahim Farzadfard
4Massachussetts Institute of Technology, Cambridge, MA
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Timothy K. Lu
4Massachussetts Institute of Technology, Cambridge, MA
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Seth L. Shipman
2University of California, San Francisco, San Francisco, CA, USA
5Gladstone Institutes, San Francisco, CA, USA
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George M. Church
1Harvard University, Boston, MA, USA
4Massachussetts Institute of Technology, Cambridge, MA
6Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, MA
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Abstract

Tremendous genetic variation exists in nature, but our ability to create and characterize individual genetic variants remains far more limited in scale. Likewise, engineering proteins and phenotypes requires the introduction of synthetic variants, but design of variants outpaces experimental measurement of variant effect. Here, we optimize efficient and continuous generation of precise genomic edits in Escherichia coli, via in-vivo production of single-stranded DNA by the targeted reverse-transcription activity of retrons. Greater than 90% editing efficiency can be obtained using this method, enabling multiplexed applications. We introduce Retron Library Recombineering (RLR), a system for high-throughput screens of variants, wherein the association of introduced edits with their retron elements enables a targeted deep sequencing phenotypic output. We use RLR for pooled, quantitative phenotyping of synthesized variants, characterizing antibiotic resistance alleles. We also perform RLR using sheared genomic DNA of an evolved bacterium, experimentally querying millions of sequences for antibiotic resistance variants. In doing so, we demonstrate that RLR is uniquely suited to utilize non-designed sources of variation. Pooled experiments using ssDNA produced in vivo thus present new avenues for exploring variation, both designed and not, across the entire genome.

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The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license.
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Posted March 06, 2020.
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High throughput functional variant screens via in-vivo production of single-stranded DNA
Max G. Schubert, Daniel B. Goodman, Timothy M. Wannier, Divjot Kaur, Fahim Farzadfard, Timothy K. Lu, Seth L. Shipman, George M. Church
bioRxiv 2020.03.05.975441; doi: https://doi.org/10.1101/2020.03.05.975441
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High throughput functional variant screens via in-vivo production of single-stranded DNA
Max G. Schubert, Daniel B. Goodman, Timothy M. Wannier, Divjot Kaur, Fahim Farzadfard, Timothy K. Lu, Seth L. Shipman, George M. Church
bioRxiv 2020.03.05.975441; doi: https://doi.org/10.1101/2020.03.05.975441

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