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Title: Simultaneous repression of multiple bacterial genes using nonrepetitive extra-long sgRNA arrays

Abstract

Engineering cellular phenotypes often requires the regulation of many genes. When using CRISPR interference, coexpressing many single-guide RNAs (sgRNAs) triggers genetic instability and phenotype loss, due to the presence of repetitive DNA sequences. We stably coexpressed 22 sgRNAs within nonrepetitive extra-long sgRNA arrays (ELSAs) to simultaneously repress up to 13 genes by up to 3,500-fold. We applied biophysical modeling, biochemical characterization and machine learning to develop toolboxes of nonrepetitive genetic parts, including 28 sgRNA handles that bind Cas9. We designed ELSAs by combining nonrepetitive genetic parts according to algorithmic rules quantifying DNA synthesis complexity, sgRNA expression, sgRNA targeting and genetic stability. Using ELSAs, we created three highly selective phenotypes in Escherichia coli, including redirecting metabolism to increase succinic acid production by 150-fold, knocking down amino acid biosynthesis to create a multi-auxotrophic strain and repressing stress responses to reduce persister cell formation by 21-fold. Finally, ELSAs enable simultaneous and stable regulation of many genes for metabolic engineering and synthetic biology applications.

Authors:
 [1];  [1];  [2];  [1]; ORCiD logo [3];  [4]; ORCiD logo [5]
  1. Pennsylvania State Univ., University Park, PA (United States). Dept of Chemical Engineering
  2. Pennsylvania State Univ., University Park, PA (United States). Dept of Biological Engineering
  3. Pennsylvania State Univ., University Park, PA (United States). Bioinformatics and Genomics
  4. Pennsylvania State Univ., University Park, PA (United States). Dept of Biochemistry and Molecular Biology
  5. Pennsylvania State Univ., University Park, PA (United States). Dept of Chemical Engineering; Pennsylvania State Univ., University Park, PA (United States). Dept of Biological Engineering; Pennsylvania State Univ., University Park, PA (United States). Bioinformatics and Genomics
Publication Date:
Research Org.:
Pennsylvania State Univ., University Park, PA (United States)
Sponsoring Org.:
USDOE Office of Science (SC), Biological and Environmental Research (BER) (SC-23). Biological Systems Science Division
OSTI Identifier:
1569832
Grant/Contract Number:  
SC0019090
Resource Type:
Accepted Manuscript
Journal Name:
Nature Biotechnology
Additional Journal Information:
Journal Name: Nature Biotechnology; Journal ID: ISSN 1087-0156
Publisher:
Springer Nature
Country of Publication:
United States
Language:
English
Subject:
60 APPLIED LIFE SCIENCES; Synthetic Biology; Metabolic Engineering; CRISPR

Citation Formats

Reis, Alexander C., Halper, Sean M., Vezeau, Grace E., Cetnar, Daniel P., Hossain, Ayaan, Clauer, Phillip R., and Salis, Howard M. Simultaneous repression of multiple bacterial genes using nonrepetitive extra-long sgRNA arrays. United States: N. p., 2019. Web. doi:10.1038/s41587-019-0286-9.
Reis, Alexander C., Halper, Sean M., Vezeau, Grace E., Cetnar, Daniel P., Hossain, Ayaan, Clauer, Phillip R., & Salis, Howard M. Simultaneous repression of multiple bacterial genes using nonrepetitive extra-long sgRNA arrays. United States. doi:10.1038/s41587-019-0286-9.
Reis, Alexander C., Halper, Sean M., Vezeau, Grace E., Cetnar, Daniel P., Hossain, Ayaan, Clauer, Phillip R., and Salis, Howard M. Mon . "Simultaneous repression of multiple bacterial genes using nonrepetitive extra-long sgRNA arrays". United States. doi:10.1038/s41587-019-0286-9.
@article{osti_1569832,
title = {Simultaneous repression of multiple bacterial genes using nonrepetitive extra-long sgRNA arrays},
author = {Reis, Alexander C. and Halper, Sean M. and Vezeau, Grace E. and Cetnar, Daniel P. and Hossain, Ayaan and Clauer, Phillip R. and Salis, Howard M.},
abstractNote = {Engineering cellular phenotypes often requires the regulation of many genes. When using CRISPR interference, coexpressing many single-guide RNAs (sgRNAs) triggers genetic instability and phenotype loss, due to the presence of repetitive DNA sequences. We stably coexpressed 22 sgRNAs within nonrepetitive extra-long sgRNA arrays (ELSAs) to simultaneously repress up to 13 genes by up to 3,500-fold. We applied biophysical modeling, biochemical characterization and machine learning to develop toolboxes of nonrepetitive genetic parts, including 28 sgRNA handles that bind Cas9. We designed ELSAs by combining nonrepetitive genetic parts according to algorithmic rules quantifying DNA synthesis complexity, sgRNA expression, sgRNA targeting and genetic stability. Using ELSAs, we created three highly selective phenotypes in Escherichia coli, including redirecting metabolism to increase succinic acid production by 150-fold, knocking down amino acid biosynthesis to create a multi-auxotrophic strain and repressing stress responses to reduce persister cell formation by 21-fold. Finally, ELSAs enable simultaneous and stable regulation of many genes for metabolic engineering and synthetic biology applications.},
doi = {10.1038/s41587-019-0286-9},
journal = {Nature Biotechnology},
number = ,
volume = ,
place = {United States},
year = {2019},
month = {10}
}

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Works referenced in this record:

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