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

doi:10.1038/s41587-019-0286-9

. 2019 Nov;37(11):1294-1301.

doi: 10.1038/s41587-019-0286-9. Epub 2019 Oct 7.

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

Alexander C Reis^#¹, Sean M Halper^#¹, Grace E Vezeau², Daniel P Cetnar¹, Ayaan Hossain³, Phillip R Clauer⁴, Howard M Salis^{5

6

7}

Affiliations

¹ Department of Chemical Engineering, Pennsylvania State University, University Park, PA, USA.
² Department of Biological Engineering, Pennsylvania State University, University Park, PA, USA.
³ Bioinformatics and Genomics, Pennsylvania State University, University Park, PA, USA.
⁴ Department of Biochemistry and Molecular Biology, Pennsylvania State University, University Park, PA, USA.
⁵ Department of Chemical Engineering, Pennsylvania State University, University Park, PA, USA. salis@psu.edu.
⁶ Department of Biological Engineering, Pennsylvania State University, University Park, PA, USA. salis@psu.edu.
⁷ Bioinformatics and Genomics, Pennsylvania State University, University Park, PA, USA. salis@psu.edu.

^# Contributed equally.

PMID: 31591552
DOI: 10.1038/s41587-019-0286-9

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

Alexander C Reis et al. Nat Biotechnol. 2019 Nov.

. 2019 Nov;37(11):1294-1301.

doi: 10.1038/s41587-019-0286-9. Epub 2019 Oct 7.

Authors

Alexander C Reis^#¹, Sean M Halper^#¹, Grace E Vezeau², Daniel P Cetnar¹, Ayaan Hossain³, Phillip R Clauer⁴, Howard M Salis^{5

6

7}

Affiliations

¹ Department of Chemical Engineering, Pennsylvania State University, University Park, PA, USA.
² Department of Biological Engineering, Pennsylvania State University, University Park, PA, USA.
³ Bioinformatics and Genomics, Pennsylvania State University, University Park, PA, USA.
⁴ Department of Biochemistry and Molecular Biology, Pennsylvania State University, University Park, PA, USA.
⁵ Department of Chemical Engineering, Pennsylvania State University, University Park, PA, USA. salis@psu.edu.
⁶ Department of Biological Engineering, Pennsylvania State University, University Park, PA, USA. salis@psu.edu.
⁷ Bioinformatics and Genomics, Pennsylvania State University, University Park, PA, USA. salis@psu.edu.

^# Contributed equally.

PMID: 31591552
DOI: 10.1038/s41587-019-0286-9

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.

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References

1. Dominguez, A. A., Lim, W. A. & Qi, L. S. Beyond editing: repurposing CRISPR–Cas9 for precision genome regulation and interrogation. Nat. Rev. Mol. Cell Biol. 17, 5–15 (2016). - PubMed
1. Barrangou, R. & Horvath, P. A decade of discovery: CRISPR functions and applications. Nat. Microbiol. 2, 17092 (2017). - PubMed
1. Halperin, S. O. et al. CRISPR-guided DNA polymerases enable diversification of all nucleotides in a tunable window. Nature 560, 248–252 (2018). - PubMed
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1. Komor, A. C., Kim, Y. B., Packer, M. S., Zuris, J. A. & Liu, D. R. Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage. Nature 533, 420–424 (2016). - PubMed - PMC

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[1] Dominguez, A. A., Lim, W. A. & Qi, L. S. Beyond editing: repurposing CRISPR–Cas9 for precision genome regulation and interrogation. Nat. Rev. Mol. Cell Biol. 17, 5–15 (2016). - PubMed

[2] Dominguez, A. A., Lim, W. A. & Qi, L. S. Beyond editing: repurposing CRISPR–Cas9 for precision genome regulation and interrogation. Nat. Rev. Mol. Cell Biol. 17, 5–15 (2016). - PubMed

[3] Barrangou, R. & Horvath, P. A decade of discovery: CRISPR functions and applications. Nat. Microbiol. 2, 17092 (2017). - PubMed

[4] Barrangou, R. & Horvath, P. A decade of discovery: CRISPR functions and applications. Nat. Microbiol. 2, 17092 (2017). - PubMed

[5] Halperin, S. O. et al. CRISPR-guided DNA polymerases enable diversification of all nucleotides in a tunable window. Nature 560, 248–252 (2018). - PubMed

[6] Halperin, S. O. et al. CRISPR-guided DNA polymerases enable diversification of all nucleotides in a tunable window. Nature 560, 248–252 (2018). - PubMed

[7] Peters, J. M. et al. Enabling genetic analysis of diverse bacteria with Mobile-CRISPRi. Nat. Microbiol. 4, 244–250 (2019). - PubMed - PMC

[8] Peters, J. M. et al. Enabling genetic analysis of diverse bacteria with Mobile-CRISPRi. Nat. Microbiol. 4, 244–250 (2019). - PubMed - PMC

[9] Komor, A. C., Kim, Y. B., Packer, M. S., Zuris, J. A. & Liu, D. R. Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage. Nature 533, 420–424 (2016). - PubMed - PMC

[10] Komor, A. C., Kim, Y. B., Packer, M. S., Zuris, J. A. & Liu, D. R. Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage. Nature 533, 420–424 (2016). - PubMed - PMC

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

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

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