Functional genomics of the rapidly replicating bacterium Vibrio natriegens by CRISPRi

• Published in: Nature microbiology Vol. 4; no. 7; pp. 1105 - 1113
• Main Authors: Lee, Henry H., Ostrov, Nili, Wong, Brandon G., Gold, Michaela A., Khalil, Ahmad S., Church, George M.
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 01-07-2019; Nature Publishing Group
• Subjects: 38/23; 38/47; 42; 45; 631/326/325; 631/553/1833; 631/553/552; 64; Bacterial Proteins - genetics; Bacterial Proteins - metabolism; BASIC BIOLOGICAL SCIENCES; Biomedical and Life Sciences; Biotechnology; Chromosome 1; Chromosome 2; Chromosomes, Bacterial - genetics; Clustered Regularly Interspaced Short Palindromic Repeats - genetics; CRISPR; CRISPR-Cas Systems; CRISPRi; Culture Media; Functional Genomics; Gene duplication; Gene Knockdown Techniques; Gene Library; Genome, Bacterial - genetics; Genomics; Infectious Diseases; Letter; Life Sciences; Medical Microbiology; Metabolic pathways; Metabolism; Microbiology; Parasitology; Vibrio - genetics; Vibrio - growth & development; Vibrio Natriegens; Virology
• ISSN: 2058-5276; 2058-5276
• DOI: 10.1038/s41564-019-0423-8

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
1
,
2
and metabolic prowess
3
,
4
. 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. 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
.
A genome-wide CRISPR interference screen of the fast-growing
Vibrio natriegens
bacterium elucidates the make-up of minimal genomes and the metabolic pathways enabling rapid bacterial replication.