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Related Concept Videos

RNA Interference01:23

RNA Interference

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RNA interference (RNAi) is a process in which a small non-coding RNA molecule blocks the post-transcriptional expression of a gene by binding to its messenger RNA (mRNA) and preventing the protein from being translated.
This process occurs naturally in cells, often through the activity of genomically-encoded microRNAs. Researchers can take advantage of this mechanism by introducing synthetic RNAs to deactivate specific genes for research or therapeutic purposes. For example, RNAi could be used...
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siRNA - Small Interfering RNAs02:30

siRNA - Small Interfering RNAs

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Small interfering RNAs, or siRNAs, are short regulatory RNA molecules that can silence genes post-transcriptionally, as well as the transcriptional level in some cases. siRNAs are important for protecting cells against viral infections and silencing transposable genetic elements.
In the cytoplasm, siRNA is processed from a double-stranded RNA, which comes from either endogenous DNA transcription or exogenous sources like a virus. This double-stranded RNA is then cleaved by the...
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CRISPR and crRNAs02:53

CRISPR and crRNAs

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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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Experimental RNAi02:15

Experimental RNAi

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RNA interference (RNAi) is a cellular mechanism that inhibits gene expression by suppressing its transcription or activating the RNA degradation process. The mechanism was discovered by Andrew Fire and Craig Mello in 1998 in plants. Today, it is observed in almost all eukaryotes, including protozoa, flies, nematodes, insects, parasites, and mammals. This precise cellular mechanism of gene silencing has been developed into a technique that provides an efficient way to identify and determine the...
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CRISPR01:59

CRISPR

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Genome editing technologies allow scientists to modify an organism’s DNA via the addition, removal, or rearrangement of genetic material at specific genomic locations. These types of techniques could potentially be used to cure genetic disorders such as hemophilia and sickle cell anemia. One popular and widely used DNA-editing research tool that could lead to safe and effective cures for genetic disorders is the CRISPR-Cas9 system. CRISPR-Cas9 stands for Clustered Regularly Interspaced...
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Small interfering RNAs (siRNA)02:30

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CRISPR Interference-Based Functional Small RNA Genomics.

Gianluca Prezza1, Alexander J Westermann2,3,4

  • 1Helmholtz Institute for RNA-based Infection Research (HIRI), Helmholtz Centre for Infection Research (HZI), Würzburg, Germany.

Methods in Molecular Biology (Clifton, N.J.)
|January 13, 2024
PubMed
Summary
This summary is machine-generated.

CRISPR interference (CRISPRi) enables functional screening of bacterial small RNAs (sRNAs) by targeting their genes. This method overcomes limitations of previous techniques, facilitating the study of sRNA roles in diverse bacteria, including gut microbiota.

Keywords:
BacteroidesCRISPRiCas12aFunctional genomicsMicrobiotaNoncoding RNAsRNA

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Area of Science:

  • Microbiology
  • Molecular Biology
  • Genomics

Background:

  • Small RNAs (sRNAs) are crucial regulators in prokaryotes, but their functions are often uncharacterized.
  • Traditional methods like transposon mutagenesis are biased against small genes, underrepresenting sRNA mutants.
  • CRISPR interference (CRISPRi) offers a length-independent approach for targeted gene knockdown.

Purpose of the Study:

  • To provide a detailed protocol for CRISPRi-based functional screening of bacterial sRNAs.
  • To demonstrate the application of CRISPRi for identifying sRNA functions using Bacteroides thetaiotaomicron as a model.
  • To enable systematic assessment of sRNA-associated phenotypes in various bacterial species.

Main Methods:

  • Development of a CRISPR interference (CRISPRi) system for targeted bacterial sRNA knockdown.
  • Design and generation of a guide RNA library targeting the intergenic sRNA repertoire.
  • Application of the CRISPRi library for fitness effect screening in Bacteroides thetaiotaomicron.

Main Results:

  • Successful implementation of CRISPRi for efficient knockdown of bacterial sRNAs.
  • Identification of fitness effects associated with specific sRNA knockdowns.
  • Demonstration of CRISPRi's suitability for unbiased screening of sRNA functions.

Conclusions:

  • CRISPR interference is a powerful and versatile tool for bacterial sRNA functional genomics.
  • This protocol facilitates the systematic study of sRNAs in underrepresented bacterial taxa, such as gut microbiota.
  • CRISPRi-based functional screening is expected to significantly advance research on bacterial sRNAs.