Repurposing the Streptococcus mutans CRISPR-Cas9 System to Understand Essential Gene Function.

PLoS Pathogens
Robert C ShieldsRobert A Burne

Abstract

A recent genome-wide screen identified ~300 essential or growth-supporting genes in the dental caries pathogen Streptococcus mutans. To be able to study these genes, we built a CRISPR interference tool around the Cas9 nuclease (Cas9Smu) encoded in the S. mutans UA159 genome. Using a xylose-inducible dead Cas9Smu with a constitutively active single-guide RNA (sgRNA), we observed titratable repression of GFP fluorescence that compared favorably to that of Streptococcus pyogenes dCas9 (Cas9Spy). We then investigated sgRNA specificity and proto-spacer adjacent motif (PAM) requirements. Interference by sgRNAs did not occur with double or triple base-pair mutations, or if single base-pair mutations were in the 3' end of the sgRNA. Bioinformatic analysis of >450 S. mutans genomes allied with in vivo assays revealed a similar PAM recognition sequence as Cas9Spy. Next, we created a comprehensive library of sgRNA plasmids that were directed at essential and growth-supporting genes. We discovered growth defects for 77% of the CRISPRi strains expressing sgRNAs. Phenotypes of CRISPRi strains, across several biological pathways, were assessed using fluorescence microscopy. A variety of cell structure anomalies were observed, including segreg...Continue Reading

References

Apr 1, 1975·Infection and Immunity·B TerleckyjG D Shockman
Jul 1, 1989·Clinical Microbiology Reviews·A L Coykendall
Apr 12, 2000·Proceedings of the National Academy of Sciences of the United States of America·S R Filipe, A Tomasz
May 25, 2002·Journal of Bacteriology·Yufang MaMichael McNeil
Jun 3, 2004·Genome Research·Gavin E CrooksSteven E Brenner
Sep 19, 2006·Nature Reviews. Microbiology·Alexei V Kazantsev, Norman R Pace
Mar 28, 2008·Molecular Microbiology·Dennis ClaessenDavid H Edwards
Jul 27, 2010·Journal of Bacteriology·Floyd E DewhirstWilliam G Wade
Aug 16, 2011·Journal of Visualized Experiments : JoVE·Imke G de JongJan-Willem Veening
Sep 29, 2011·Nature Communications·Kazuhiko NakanoTakashi Ooshima
Nov 26, 2011·Algorithms for Molecular Biology : AMB·Ronny LorenzIvo L Hofacker
Jun 30, 2012·Science·Martin JinekEmmanuelle Charpentier
Sep 7, 2012·Trends in Biotechnology·Mario JuhasGeorge M Church
Feb 9, 2013·Microbiology·José A LemosJacqueline Abranches
Mar 16, 2013·RNA Biology·Ambarish BiswasChris M Brown
Apr 2, 2013·Journal of Bacteriology·Zhiyun ChenJens Kreth
May 1, 2013·Nature Reviews. Drug Discovery·Kim Lewis
Jun 14, 2013·Nucleic Acids Research·David BikardLuciano A Marraffini
Jul 16, 2013·Environmental Microbiology·Orietta MassiddaWaldemar Vollmer
Oct 22, 2013·European Journal of Clinical Microbiology & Infectious Diseases : Official Publication of the European Society of Clinical Microbiology·W KrzyściakD Kościelniak
Dec 10, 2014·Journal of Bacteriology·M A SerbanescuD G Cvitkovitch
Feb 25, 2015·Nature·Robert HelerLuciano A Marraffini
Jun 23, 2015·Nature·Benjamin P KleinstiverJ Keith Joung
Jul 21, 2015·Journal of Biotechnology·Emily M AndersonAnja van Brabant Smith
Mar 16, 2016·FEMS Microbiology Reviews·Michel-Yves MistouNina M van Sorge
Apr 5, 2016·Molecular Cell·Ryan T LeenayChase L Beisel
May 12, 2017·Molecular Systems Biology·Xue LiuJan-Willem Veening
May 13, 2017·Proceedings of the National Academy of Sciences of the United States of America·Evan A BoyleWilliam J Greenleaf
May 26, 2017·Nature Reviews. Disease Primers·Nigel B PittsAmid Ismail
Feb 12, 2016·Synthetic and Systems Biotechnology·Kai BlinSang Yup Lee
Feb 10, 2018·Cell Reports·Misha KleinMartin Depken

❮ Previous
Next ❯

Citations

Dec 8, 2020·Current Opinion in Microbiology·Horia TodorCarol A Gross
Jan 24, 2021·Nature Communications·Daphne Collias, Chase L Beisel

❮ Previous
Next ❯

Methods Mentioned

BETA
Tn-seq
gene knockdown
phosphotransferase
RNA-seq
fluorescence microscopy
two-hybrid
transmission electron microscopy
electron microscopy
PCR

Software Mentioned

CRISPRTarget
ViennaRNA
BLASTp
RNAfold
GraphPad Prism
CRISPRone
ImageJ
WebLogo
GraphPad
BLASTn

Related Concepts

Related Feeds

CRISPR Ribonucleases Deactivation

CRISPR-Cas system enables the editing of genes to create or correct mutations. This feed focuses on mechanisms that underlie deactivation of CRISPR ribonucleases. Here is the latest research.

CRISPR (general)

Clustered regularly interspaced short palindromic repeats (CRISPR) are DNA sequences in the genome that are recognized and cleaved by CRISPR-associated proteins (Cas). CRISPR-Cas system enables the editing of genes to create or correct mutations. Discover the latest research on CRISPR here.