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Title: Improved bacterial recombineering by parallelized protein discovery

Abstract

Exploiting bacteriophage-derived homologous recombination processes has offered precise, multiplex editing of microbial genomes and the construction of billions of customized genetic variants in a single day. The techniques that enable this, multiplex automated genome engineering (MAGE) and directed evolution with random genomic mutations (DIvERGE), are however, currently limited to a handful of microorganisms for which single-stranded DNA-annealing proteins (SSAPs) that promote efficient recombineering have been identified. Thus, to enable genome-scale engineering in new hosts, efficient SSAPs must first be found. Here we present a high-throughput method for SSAP discovery that we call “serial enrichment for efficient recombineering” (SEER). By performing SEER inEscherichia colito screen hundreds of putative SSAPs, we identify highly active variants PapRecT and CspRecT. CspRecT increases the efficiency of single-locus editing to as high as 50% and improves multiplex editing by 5- to 10-fold inE. coli, while PapRecT enables efficient recombineering inPseudomonas aeruginosa, a concerning human pathogen. CspRecT and PapRecT are also active in other, clinically and biotechnologically relevant enterobacteria. We envision that the deployment of SEER in new species will pave the way toward pooled interrogation of genotype-to-phenotype relationships in previously intractable bacteria.

Authors:
ORCiD logo; ORCiD logo; ; ; ; ORCiD logo; ; ; ORCiD logo; ; ; ORCiD logo
Publication Date:
Research Org.:
Harvard Medical School, Boston, MA (United States)
Sponsoring Org.:
USDOE Office of Science and Technology (ST); USDOE Office of Science (SC), Biological and Environmental Research (BER); European Research Council (ERC)
OSTI Identifier:
1631183
Alternate Identifier(s):
OSTI ID: 1631102
Grant/Contract Number:  
FG02-02ER63445
Resource Type:
Published Article
Journal Name:
Proceedings of the National Academy of Sciences of the United States of America
Additional Journal Information:
Journal Name: Proceedings of the National Academy of Sciences of the United States of America; Journal ID: ISSN 0027-8424
Publisher:
National Academy of Sciences
Country of Publication:
United States
Language:
English
Subject:
60 APPLIED LIFE SCIENCES; recombineering; genome editing; multiplex automated genome engineering

Citation Formats

Wannier, Timothy M., Nyerges, Akos, Kuchwara, Helene M., Czikkely, Márton, Balogh, Dávid, Filsinger, Gabriel T., Borders, Nathaniel C., Gregg, Christopher J., Lajoie, Marc J., Rios, Xavier, Pál, Csaba, and Church, George M. Improved bacterial recombineering by parallelized protein discovery. United States: N. p., 2020. Web. https://doi.org/10.1073/pnas.2001588117.
Wannier, Timothy M., Nyerges, Akos, Kuchwara, Helene M., Czikkely, Márton, Balogh, Dávid, Filsinger, Gabriel T., Borders, Nathaniel C., Gregg, Christopher J., Lajoie, Marc J., Rios, Xavier, Pál, Csaba, & Church, George M. Improved bacterial recombineering by parallelized protein discovery. United States. https://doi.org/10.1073/pnas.2001588117
Wannier, Timothy M., Nyerges, Akos, Kuchwara, Helene M., Czikkely, Márton, Balogh, Dávid, Filsinger, Gabriel T., Borders, Nathaniel C., Gregg, Christopher J., Lajoie, Marc J., Rios, Xavier, Pál, Csaba, and Church, George M. Thu . "Improved bacterial recombineering by parallelized protein discovery". United States. https://doi.org/10.1073/pnas.2001588117.
@article{osti_1631183,
title = {Improved bacterial recombineering by parallelized protein discovery},
author = {Wannier, Timothy M. and Nyerges, Akos and Kuchwara, Helene M. and Czikkely, Márton and Balogh, Dávid and Filsinger, Gabriel T. and Borders, Nathaniel C. and Gregg, Christopher J. and Lajoie, Marc J. and Rios, Xavier and Pál, Csaba and Church, George M.},
abstractNote = {Exploiting bacteriophage-derived homologous recombination processes has offered precise, multiplex editing of microbial genomes and the construction of billions of customized genetic variants in a single day. The techniques that enable this, multiplex automated genome engineering (MAGE) and directed evolution with random genomic mutations (DIvERGE), are however, currently limited to a handful of microorganisms for which single-stranded DNA-annealing proteins (SSAPs) that promote efficient recombineering have been identified. Thus, to enable genome-scale engineering in new hosts, efficient SSAPs must first be found. Here we present a high-throughput method for SSAP discovery that we call “serial enrichment for efficient recombineering” (SEER). By performing SEER inEscherichia colito screen hundreds of putative SSAPs, we identify highly active variants PapRecT and CspRecT. CspRecT increases the efficiency of single-locus editing to as high as 50% and improves multiplex editing by 5- to 10-fold inE. coli, while PapRecT enables efficient recombineering inPseudomonas aeruginosa, a concerning human pathogen. CspRecT and PapRecT are also active in other, clinically and biotechnologically relevant enterobacteria. We envision that the deployment of SEER in new species will pave the way toward pooled interrogation of genotype-to-phenotype relationships in previously intractable bacteria.},
doi = {10.1073/pnas.2001588117},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
number = ,
volume = ,
place = {United States},
year = {2020},
month = {5}
}

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https://doi.org/10.1073/pnas.2001588117

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