A Comprehensive, CRISPR-based Functional Analysis of Essential Genes in Bacteria

Jason M Peters; Alexandre Colavin; Handuo Shi; Tomasz L Czarny; Matthew H Larson; Spencer Wong; John S Hawkins; Candy H S Lu; Byoung-Mo Koo; Elizabeth Marta; Anthony L Shiver; Evan H Whitehead; Jonathan S Weissman; Eric D Brown; Lei S Qi; Kerwyn Casey Huang; Carol A Gross

doi:10.1016/j.cell.2016.05.003

A Comprehensive, CRISPR-based Functional Analysis of Essential Genes in Bacteria

Cell. 2016 Jun 2;165(6):1493-1506. doi: 10.1016/j.cell.2016.05.003. Epub 2016 May 26.

Authors

Jason M Peters¹, Alexandre Colavin², Handuo Shi³, Tomasz L Czarny⁴, Matthew H Larson⁵, Spencer Wong⁶, John S Hawkins⁷, Candy H S Lu¹, Byoung-Mo Koo¹, Elizabeth Marta¹, Anthony L Shiver⁷, Evan H Whitehead⁸, Jonathan S Weissman⁹, Eric D Brown⁴, Lei S Qi¹⁰, Kerwyn Casey Huang¹¹, Carol A Gross¹²

Affiliations

¹ Department of Microbiology and Immunology, University of California, San Francisco, San Francisco, CA 94158, USA.
² Biophysics Program, Stanford University, Stanford, CA 94305, USA.
³ Department of Bioengineering, Stanford University, Stanford, CA 94305, USA.
⁴ Department of Biochemistry and Biomedical Sciences, McMaster University, Hamilton, ON L8S 3Z5, Canada; Michael G. DeGroote Institute of Infectious Disease Research, McMaster University, Hamilton, ON L8N 3Z5, Canada.
⁵ Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, CA 94158, USA; Howard Hughes Medical Institute, University of California, San Francisco, San Francisco, CA 94158, USA; California Institute for Quantitative Biomedical Research, San Francisco, CA 94158, USA; Center for RNA Systems Biology, University of California, Berkeley, Berkeley, CA 94720, USA.
⁶ Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, CA 94158, USA.
⁷ Department of Microbiology and Immunology, University of California, San Francisco, San Francisco, CA 94158, USA; Biophysics Graduate Program, University of California, San Francisco, San Francisco, CA 94158, USA.
⁸ Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, CA 94158, USA; California Institute for Quantitative Biomedical Research, San Francisco, CA 94158, USA; UCSF Center for Systems and Synthetic Biology, University of California, San Francisco, San Francisco, CA 94158, USA.
⁹ Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, CA 94158, USA; Howard Hughes Medical Institute, University of California, San Francisco, San Francisco, CA 94158, USA; California Institute for Quantitative Biomedical Research, San Francisco, CA 94158, USA; Center for RNA Systems Biology, University of California, Berkeley, Berkeley, CA 94720, USA; UCSF Center for Systems and Synthetic Biology, University of California, San Francisco, San Francisco, CA 94158, USA.
¹⁰ Department of Bioengineering, Stanford University, Stanford, CA 94305, USA; Department of Chemical and Systems Biology, Stanford University, Stanford, CA 94305, USA; ChEM-H, Stanford University, Stanford, CA 94305, USA. Electronic address: stanley.qi@stanford.edu.
¹¹ Biophysics Program, Stanford University, Stanford, CA 94305, USA; Department of Bioengineering, Stanford University, Stanford, CA 94305, USA; School of Medicine, Department of Microbiology and Immunology, Stanford University, Stanford, CA 94305, USA. Electronic address: kchuang@stanford.edu.
¹² Department of Microbiology and Immunology, University of California, San Francisco, San Francisco, CA 94158, USA. Electronic address: cgrossucsf@gmail.com.

Abstract

Essential gene functions underpin the core reactions required for cell viability, but their contributions and relationships are poorly studied in vivo. Using CRISPR interference, we created knockdowns of every essential gene in Bacillus subtilis and probed their phenotypes. Our high-confidence essential gene network, established using chemical genomics, showed extensive interconnections among distantly related processes and identified modes of action for uncharacterized antibiotics. Importantly, mild knockdown of essential gene functions significantly reduced stationary-phase survival without affecting maximal growth rate, suggesting that essential protein levels are set to maximize outgrowth from stationary phase. Finally, high-throughput microscopy indicated that cell morphology is relatively insensitive to mild knockdown but profoundly affected by depletion of gene function, revealing intimate connections between cell growth and shape. Our results provide a framework for systematic investigation of essential gene functions in vivo broadly applicable to diverse microorganisms and amenable to comparative analysis.

Published by Elsevier Inc.

Publication types

Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't
Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

Bacillus subtilis / genetics*
CRISPR-Cas Systems
Gene Knockdown Techniques
Gene Library
Gene Regulatory Networks
Genes, Bacterial*
Genes, Essential*
Molecular Targeted Therapy

Abstract

Publication types

MeSH terms

Grants and funding