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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, co-expressing many single-guide RNAs (sgRNAs) triggers genetic instability and phenotype loss, due to the presence of repetitive DNA sequences. We stably co-expressed 22 sgRNAs within non-repetitive extra-long sgRNA arrays (ELSAs) to simultaneously repress up to 13 genes by up to 3500-fold. We apply biophysical modeling, biochemical characterization, and machine learning to develop toolboxes of non-repetitive genetic parts, including 28 sgRNA handles that bind Cas9. We design ELSAs by combining non-repetitive 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. ELSAs enable simultaneous and stable regulation of many genes for metabolic engineering and synthetic biology applications.

Authors:
 [1];  [1];  [1];  [1]; ORCiD logo [1];  [1]; ORCiD logo [1]
  1. Pennsylvania State Univ. University Park, PA (United States)
Publication Date:
Research Org.:
Pennsylvania State Univ., University Park, PA (United States)
Sponsoring Org.:
USDOE Office of Science (SC), Biological and Environmental Research (BER). Biological Systems Science Division; US Air Force Office of Scientific Research (AFOSR); Defense Advanced Research Projects Agency (DARPA)
OSTI Identifier:
1569832
Alternate Identifier(s):
OSTI ID: 1593337
Grant/Contract Number:  
SC0019090; FA9550-14-1-0089; CBET-1253641; FA8750-17-C-0254
Resource Type:
Accepted Manuscript
Journal Name:
Nature Biotechnology
Additional Journal Information:
Journal Volume: 37; Journal Issue: 11; 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; synthetic biology; CRISPR/Cas9

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. https://www.osti.gov/servlets/purl/1569832.
@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, co-expressing many single-guide RNAs (sgRNAs) triggers genetic instability and phenotype loss, due to the presence of repetitive DNA sequences. We stably co-expressed 22 sgRNAs within non-repetitive extra-long sgRNA arrays (ELSAs) to simultaneously repress up to 13 genes by up to 3500-fold. We apply biophysical modeling, biochemical characterization, and machine learning to develop toolboxes of non-repetitive genetic parts, including 28 sgRNA handles that bind Cas9. We design ELSAs by combining non-repetitive 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. 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 = 11,
volume = 37,
place = {United States},
year = {2019},
month = {10}
}

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