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.

PubMed Disclaimer

Cited by

EcCas6e-based antisense crRNA for gene repression and RNA editing in microorganisms.
Li M, Cai Z, Song S, Yue X, Lu W, Rao S, Zhang C, Xue C. Li M, et al. Nucleic Acids Res. 2024 Aug 12;52(14):8628-8642. doi: 10.1093/nar/gkae612. Nucleic Acids Res. 2024. PMID: 38994565 Free PMC article.
Tuning a high performing multiplexed-CRISPRi Pseudomonas putida strain to further enhance indigoidine production.
Czajka JJ, Banerjee D, Eng T, Menasalvas J, Yan C, Munoz NM, Poirier BC, Kim YM, Baker SE, Tang YJ, Mukhopadhyay A. Czajka JJ, et al. Metab Eng Commun. 2022 Sep 13;15:e00206. doi: 10.1016/j.mec.2022.e00206. eCollection 2022 Dec. Metab Eng Commun. 2022. PMID: 36158112 Free PMC article.
Comprehensive double-mutant analysis of the Bacillus subtilis envelope using double-CRISPRi.
Koo BM, Todor H, Sun J, van Gestel J, Hawkins JS, Hearne CC, Banta AB, Huang KC, Peters JM, Gross CA. Koo BM, et al. bioRxiv [Preprint]. 2024 Aug 16:2024.08.14.608006. doi: 10.1101/2024.08.14.608006. bioRxiv. 2024. PMID: 39185233 Free PMC article. Preprint.
Mismatch-CRISPRi Reveals the Co-varying Expression-Fitness Relationships of Essential Genes in Escherichia coli and Bacillus subtilis.
Hawkins JS, Silvis MR, Koo BM, Peters JM, Osadnik H, Jost M, Hearne CC, Weissman JS, Todor H, Gross CA. Hawkins JS, et al. Cell Syst. 2020 Nov 18;11(5):523-535.e9. doi: 10.1016/j.cels.2020.09.009. Epub 2020 Oct 19. Cell Syst. 2020. PMID: 33080209 Free PMC article.
Comparative optimization of combinatorial CRISPR screens.
Li R, Klingbeil O, Monducci D, Young MJ, Rodriguez DJ, Bayyat Z, Dempster JM, Kesar D, Yang X, Zamanighomi M, Vakoc CR, Ito T, Sellers WR. Li R, et al. Nat Commun. 2022 May 5;13(1):2469. doi: 10.1038/s41467-022-30196-9. Nat Commun. 2022. PMID: 35513429 Free PMC article.

See all "Cited by" articles

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

Publication types

Actions
Actions

MeSH terms

Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions

Substances

Actions
Actions
Actions
Actions
Actions

LinkOut - more resources

Full Text Sources
- Nature Publishing Group
Other Literature Sources
- The Lens - Patent Citations Database

[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

Save citation to file

Email citation

Add to Collections

Add to My Bibliography

Your saved search

Create a file for external citation management software

Your RSS Feed

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

Affiliations

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

Authors

Affiliations

Abstract

Similar articles

Cited by

References

Publication types

MeSH terms

Substances

LinkOut - more resources

Full Text Sources

Other Literature Sources

Abstract

Similar articles

Cited by

References

Publication types

MeSH terms

Substances

Related information

LinkOut - more resources

Full Text Sources

Other Literature Sources