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.

doi:10.1038/s41587-019-0286-9

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

Journal Article · Mon Oct 07 00:00:00 EDT 2019 · Nature Biotechnology

DOI:https://doi.org/10.1038/s41587-019-0286-9· OSTI ID:1569832

Reis, Alexander C. ^[1]; Halper, Sean M. ^[2]; Vezeau, Grace E. ^[2]; Cetnar, Daniel P. ^[2]; ^[2]; Clauer, Phillip R. ^[2]; ^[2]

Pennsylvania State Univ. University Park, PA (United States); Penn State University
Pennsylvania State Univ. University Park, PA (United States)

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.

View Accepted Manuscript (DOE)

Research Organization:: Pennsylvania State Univ., University Park, PA (United States)

Sponsoring Organization:: Defense Advanced Research Projects Agency (DARPA); US Air Force Office of Scientific Research (AFOSR); USDOE Office of Science (SC), Biological and Environmental Research (BER) (SC-23). Biological Systems Science Division

Grant/Contract Number:: SC0019090

OSTI ID:: 1569832

Alternate ID(s):: OSTI ID: 1593337

Journal Information:: Nature Biotechnology, Journal Name: Nature Biotechnology Journal Issue: 11 Vol. 37; ISSN 1087-0156

Publisher:: Springer NatureCopyright Statement

Country of Publication:: United States

Language:: English

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