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

Alexander C. Reis, Sean M. Halper, Grace E. Vezeau, Daniel P. Cetnar, Ayaan Hossain, Phillip R. Clauer, Howard M. Salis

Research output: Contribution to journalLetter

1 Citation (Scopus)

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. ELSAs enable simultaneous and stable regulation of many genes for metabolic engineering and synthetic biology applications.

Original languageEnglish (US)
Pages (from-to)1294-1301
Number of pages8
JournalNature Biotechnology
Volume37
Issue number11
DOIs
StatePublished - Nov 1 2019

Fingerprint

Bacterial Genes
Guide RNA
Genes
RNA
Phenotype
Clustered Regularly Interspaced Short Palindromic Repeats
Synthetic Biology
Metabolic engineering
Cell Engineering
Metabolic Engineering
Nucleic Acid Repetitive Sequences
DNA sequences
Biosynthesis
Succinic Acid
Metabolism
Escherichia coli
Learning systems
Amino acids
DNA
Amino Acids

All Science Journal Classification (ASJC) codes

  • Biotechnology
  • Bioengineering
  • Applied Microbiology and Biotechnology
  • Molecular Medicine
  • Biomedical Engineering

Cite this

Reis, A. C., Halper, S. M., Vezeau, G. E., Cetnar, D. P., Hossain, A., Clauer, P. R., & Salis, H. M. (2019). Simultaneous repression of multiple bacterial genes using nonrepetitive extra-long sgRNA arrays. Nature Biotechnology, 37(11), 1294-1301. https://doi.org/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. In: Nature Biotechnology. 2019 ; Vol. 37, No. 11. pp. 1294-1301.
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Reis, AC, Halper, SM, Vezeau, GE, Cetnar, DP, Hossain, A, Clauer, PR & Salis, HM 2019, 'Simultaneous repression of multiple bacterial genes using nonrepetitive extra-long sgRNA arrays', Nature Biotechnology, vol. 37, no. 11, pp. 1294-1301. https://doi.org/10.1038/s41587-019-0286-9

Simultaneous repression of multiple bacterial genes using nonrepetitive extra-long sgRNA arrays. / Reis, Alexander C.; Halper, Sean M.; Vezeau, Grace E.; Cetnar, Daniel P.; Hossain, Ayaan; Clauer, Phillip R.; Salis, Howard M.

In: Nature Biotechnology, Vol. 37, No. 11, 01.11.2019, p. 1294-1301.

Research output: Contribution to journalLetter

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