Construction of an Arrayed CRISPRi Library As a Resource for Essential Gene Function Studies in

Overview

Journal Microbiol Spectr

Specialty Microbiology

Date 2023 Dec 6

PMID 38054713

Authors

Jackson St Pierre

Justin Roberts

Mohammad A Alam

Robert C Shields

Affiliations

Soon will be listed here.

Abstract

The construction of arrayed mutant libraries has advanced the field of bacterial genetics by allowing researchers to more efficiently study the exact function and importance of encoded genes. In this study, we constructed an arrayed clustered regularly interspaced short palindromic repeats interference (CRISPRi) library, known as rrayed CRISRi (SNAP), as a resource to study >250 essential and growth-supporting genes in . SNAP will be made available to the research community, and we anticipate that its distribution will lead to high-quality, high-throughput, and reproducible studies of essential genes.

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References

Liao Y, Chen J, Brandt B, Zhu Y, Li J, van Loveren C . Identification and functional analysis of genome mutations in a fluoride-resistant Streptococcus mutans strain. PLoS One. 2015; 10(4):e0122630. PMC: 4391945. DOI: 10.1371/journal.pone.0122630. View

Anglada-Girotto M, Handschin G, Ortmayr K, Campos A, Gillet L, Manfredi P . Combining CRISPRi and metabolomics for functional annotation of compound libraries. Nat Chem Biol. 2022; 18(5):482-491. PMC: 7612681. DOI: 10.1038/s41589-022-00970-3. View

Hoelscher G, Hudson M . Characterization of an unusual fluoride-resistant Streptococcus mutans isolate. Curr Microbiol. 1996; 32(3):156-61. DOI: 10.1007/s002849900028. View

Baba T, Ara T, Hasegawa M, Takai Y, Okumura Y, Baba M . Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection. Mol Syst Biol. 2006; 2:2006.0008. PMC: 1681482. DOI: 10.1038/msb4100050. View

Willett J, Ji M, Dunny G . Exploiting biofilm phenotypes for functional characterization of hypothetical genes in . NPJ Biofilms Microbiomes. 2019; 5(1):23. PMC: 6753144. DOI: 10.1038/s41522-019-0099-0. View

Jacobs M, Alwood A, Thaipisuttikul I, Spencer D, Haugen E, Ernst S . Comprehensive transposon mutant library of Pseudomonas aeruginosa. Proc Natl Acad Sci U S A. 2003; 100(24):14339-44. PMC: 283593. DOI: 10.1073/pnas.2036282100. View

Lemos J, Palmer S, Zeng L, Wen Z, Kajfasz J, Freires I . The Biology of . Microbiol Spectr. 2019; 7(1). PMC: 6615571. DOI: 10.1128/microbiolspec.GPP3-0051-2018. View

de Bakker V, Liu X, Bravo A, Veening J . CRISPRi-seq for genome-wide fitness quantification in bacteria. Nat Protoc. 2022; 17(2):252-281. DOI: 10.1038/s41596-021-00639-6. View

Yu J, Wang Y, Han D, Cao W, Zheng L, Xie Z . Identification of Streptococcus mutans genes involved in fluoride resistance by screening of a transposon mutant library. Mol Oral Microbiol. 2020; 35(6):260-270. DOI: 10.1111/omi.12316. View

10.

Xie Z, Qi F, Merritt J . Development of a tunable wide-range gene induction system useful for the study of streptococcal toxin-antitoxin systems. Appl Environ Microbiol. 2013; 79(20):6375-84. PMC: 3811191. DOI: 10.1128/AEM.02320-13. View

11.

Silvis M, Rajendram M, Shi H, Osadnik H, Gray A, Cesar S . Morphological and Transcriptional Responses to CRISPRi Knockdown of Essential Genes in Escherichia coli. mBio. 2021; 12(5):e0256121. PMC: 8510551. DOI: 10.1128/mBio.02561-21. View

12.

Rostain W, Grebert T, Vyhovskyi D, Thiel Pizarro P, Tshinsele-Van Bellingen G, Cui L . Cas9 off-target binding to the promoter of bacterial genes leads to silencing and toxicity. Nucleic Acids Res. 2023; 51(7):3485-3496. PMC: 10123097. DOI: 10.1093/nar/gkad170. View

13.

Roy S, Kc H, Roberts J, Hastings J, Gilmore D, Shields R . Development and Antibacterial Properties of 4-[4-(Anilinomethyl)-3-phenylpyrazol-1-yl]benzoic Acid Derivatives as Fatty Acid Biosynthesis Inhibitors. J Med Chem. 2023; 66(19):13622-13645. PMC: 10591900. DOI: 10.1021/acs.jmedchem.3c00969. View

14.

Liao Y, Yang J, Brandt B, Li J, Crielaard W, van Loveren C . Genetic Loci Associated With Fluoride Resistance in . Front Microbiol. 2019; 9:3093. PMC: 6297193. DOI: 10.3389/fmicb.2018.03093. View

15.

de Wet T, Winkler K, Mhlanga M, Mizrahi V, Warner D . Arrayed CRISPRi and quantitative imaging describe the morphotypic landscape of essential mycobacterial genes. Elife. 2020; 9. PMC: 7647400. DOI: 10.7554/eLife.60083. View

16.

Dale J, Beckman K, Willett J, Nilson J, Palani N, Baller J . Comprehensive Functional Analysis of the Enterococcus faecalis Core Genome Using an Ordered, Sequence-Defined Collection of Insertional Mutations in Strain OG1RF. mSystems. 2018; 3(5). PMC: 6134198. DOI: 10.1128/mSystems.00062-18. View

17.

Blin K, Pedersen L, Weber T, Lee S . CRISPy-web: An online resource to design sgRNAs for CRISPR applications. Synth Syst Biotechnol. 2017; 1(2):118-121. PMC: 5640694. DOI: 10.1016/j.synbio.2016.01.003. View

18.

Larson M, Gilbert L, Wang X, Lim W, Weissman J, Qi L . CRISPR interference (CRISPRi) for sequence-specific control of gene expression. Nat Protoc. 2013; 8(11):2180-96. PMC: 3922765. DOI: 10.1038/nprot.2013.132. View

19.

King S, Quick A, King K, Walker A, Shields R . Activation of TnSmu1, an integrative and conjugative element, by an ImmR-like transcriptional regulator in . Microbiology (Reading). 2022; 168(10). PMC: 10233336. DOI: 10.1099/mic.0.001254. View

20.

Kajfasz J, Zuber P, Ganguly T, Abranches J, Lemos J . Increased Oxidative Stress Tolerance of a Spontaneously Occurring Gene Mutation in Streptococcus mutans UA159. J Bacteriol. 2021; 203(8). PMC: 8088507. DOI: 10.1128/JB.00535-20. View