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Bacteria and archaea are susceptible to viral infections just like eukaryotes; therefore, they have developed a unique adaptive immune system to protect themselves. Clustered regularly interspaced short palindromic repeats and CRISPR-associated proteins (CRISPR-Cas) are present in more than 45% of known bacteria and 90% of known archaea.
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Application of CRISPR Interference CRISPRi for Gene Silencing in Pathogenic Species of Leptospira
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Transcriptional Knockdown in Pneumococci Using CRISPR Interference.

Morten Kjos1

  • 1Faculty of Chemistry, Biotechnology and Food Science, Norwegian University of Life Sciences, Ås, Norway. morten.kjos@nmbu.no.

Methods in Molecular Biology (Clifton, N.J.)
|April 1, 2019
PubMed
Summary
This summary is machine-generated.

This study introduces a CRISPR interference (CRISPRi) method for Streptococcus pneumoniae, enabling targeted gene knockdown. The protocol details designing single guide RNAs (sgRNAs) for precise gene silencing in this bacterium.

Keywords:
CRISPRiInverse PCRKnockdowndCas9sgRNA

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Area of Science:

  • Molecular Biology
  • Microbiology
  • Gene Regulation

Background:

  • CRISPR interference (CRISPRi) offers sequence-specific gene knockdown.
  • This technology is newly adapted for the bacterium Streptococcus pneumoniae.
  • CRISPRi utilizes a catalytically inactive Cas9 protein (dCas9) and a single guide RNA (sgRNA).

Purpose of the Study:

  • To provide a protocol for designing sgRNAs for CRISPRi in S. pneumoniae.
  • To outline the construction of CRISPRi strains in S. pneumoniae.
  • To enable targeted gene expression knockdown in S. pneumoniae.

Main Methods:

  • Designing sgRNAs with a 20 bp targeting sequence for gene specificity.
  • Coexpressing dCas9 and sgRNA in S. pneumoniae.
  • Utilizing established vectors for CRISPRi strain construction.

Main Results:

  • A functional CRISPRi system for S. pneumoniae was established.
  • The protocol facilitates the targeting of specific genes for knockdown.
  • The method allows for the facile modification of sgRNAs to target new genes.

Conclusions:

  • The developed protocol enables efficient and specific gene knockdown in S. pneumoniae.
  • This CRISPRi system is a valuable tool for studying gene function in S. pneumoniae.
  • The protocol is adaptable for targeting diverse genes within the S. pneumoniae genome.