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Title: Functional genomics of the rapidly replicating bacterium Vibrio natriegens by CRISPRi

Abstract

We present that the fast-growing Gram-negative bacterium Vibrio natriegens is an attractive microbial system for molecular biology and biotechnology due to its remarkably short generation time and metabolic prowess. However, efforts to uncover and utilize the mechanisms underlying its rapid growth are hampered by the scarcity of functional genomic data. Here, we develop a pooled genome-wide clustered regularly interspaced short palindromic repeats (CRISPR) interference (CRISPRi) screen to identify a minimal set of genes required for rapid wild-type growth. Targeting 4,565 (99.7%) of predicted protein-coding genes, our screen uncovered core genes comprising putative essential and growth-supporting genes that are enriched for respiratory pathways. We found that 96% of core genes were located on the larger chromosome 1, with growth-neutral duplicates of core genes located primarily on chromosome 2. Lastly, our screen also refines metabolic pathway annotations by distinguishing functional biosynthetic enzymes from those predicted on the basis of comparative genomics. Taken together, this work provides a broadly applicable platform for high-throughput functional genomics to accelerate biological studies and engineering of V. natriegens.

Authors:
ORCiD logo [1]; ORCiD logo [1]; ORCiD logo [2];  [1]; ORCiD logo [3]; ORCiD logo [4]
  1. Harvard Medical School, Boston, MA (United States)
  2. Boston Univ., MA (United States)
  3. Boston Univ., MA (United States); Harvard Univ., Boston, MA (United States)
  4. Harvard Medical School, Boston, MA (United States); Harvard Univ., Boston, MA (United States)
Publication Date:
Research Org.:
Harvard Medical School, Boston, MA (United States)
Sponsoring Org.:
USDOE Office of Science (SC), Biological and Environmental Research (BER) (SC-23). Biological Systems Science Division
Contributing Org.:
AWS Cloud Credits for Research
OSTI Identifier:
1511570
Grant/Contract Number:  
FG02-02ER63445
Resource Type:
Journal Article: Accepted Manuscript
Journal Name:
Nature Microbiology
Additional Journal Information:
Related Information: Supplementary Information: https://www.nature.com/articles/s41564-019-0423-8#Sec23Genome Sequences: https://www.ncbi.nlm.nih.gov/nuccore/CP009977,CP009978gRNA count data: https://www.ncbi.nlm.nih.gov/bioproject/PRJNA511728transcriptome data: https://www.ncbi.nlm.nih.gov/bioproject/?term=GSE126544; Journal ID: ISSN 2058-5276
Publisher:
Nature Publishing Group
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; Functional Genomics; Vibrio Natriegens; CRISPRi

Citation Formats

Lee, Henry H., Ostrov, Nili, Wong, Brandon G., Gold, Michaela A., Khalil, Ahmad S., and Church, George M. Functional genomics of the rapidly replicating bacterium Vibrio natriegens by CRISPRi. United States: N. p., 2019. Web. doi:10.1038/s41564-019-0423-8.
Lee, Henry H., Ostrov, Nili, Wong, Brandon G., Gold, Michaela A., Khalil, Ahmad S., & Church, George M. Functional genomics of the rapidly replicating bacterium Vibrio natriegens by CRISPRi. United States. doi:10.1038/s41564-019-0423-8.
Lee, Henry H., Ostrov, Nili, Wong, Brandon G., Gold, Michaela A., Khalil, Ahmad S., and Church, George M. Mon . "Functional genomics of the rapidly replicating bacterium Vibrio natriegens by CRISPRi". United States. doi:10.1038/s41564-019-0423-8.
@article{osti_1511570,
title = {Functional genomics of the rapidly replicating bacterium Vibrio natriegens by CRISPRi},
author = {Lee, Henry H. and Ostrov, Nili and Wong, Brandon G. and Gold, Michaela A. and Khalil, Ahmad S. and Church, George M.},
abstractNote = {We present that the fast-growing Gram-negative bacterium Vibrio natriegens is an attractive microbial system for molecular biology and biotechnology due to its remarkably short generation time and metabolic prowess. However, efforts to uncover and utilize the mechanisms underlying its rapid growth are hampered by the scarcity of functional genomic data. Here, we develop a pooled genome-wide clustered regularly interspaced short palindromic repeats (CRISPR) interference (CRISPRi) screen to identify a minimal set of genes required for rapid wild-type growth. Targeting 4,565 (99.7%) of predicted protein-coding genes, our screen uncovered core genes comprising putative essential and growth-supporting genes that are enriched for respiratory pathways. We found that 96% of core genes were located on the larger chromosome 1, with growth-neutral duplicates of core genes located primarily on chromosome 2. Lastly, our screen also refines metabolic pathway annotations by distinguishing functional biosynthetic enzymes from those predicted on the basis of comparative genomics. Taken together, this work provides a broadly applicable platform for high-throughput functional genomics to accelerate biological studies and engineering of V. natriegens.},
doi = {10.1038/s41564-019-0423-8},
journal = {Nature Microbiology},
issn = {2058-5276},
number = ,
volume = ,
place = {United States},
year = {2019},
month = {4}
}

Journal Article:
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Works referenced in this record:

Repurposing CRISPR as an RNA-Guided Platform for Sequence-Specific Control of Gene Expression
journal, February 2013