A CRISPRi screen in E. coli reveals sequence-specific toxicity of dCas9

Related Experiment Video

Updated: Feb 10, 2026

Application of CRISPR Interference CRISPRi for Gene Silencing in Pathogenic Species of Leptospira
14:49

Application of CRISPR Interference CRISPRi for Gene Silencing in Pathogenic Species of Leptospira

Published on: August 14, 2021

5.7K

Lun Cui1, Antoine Vigouroux1, François Rousset1,2

  • 1Synthetic Biology Group, Department of Microbiology, Institut Pasteur, Paris, 75015, France.

Nature Communications
|May 17, 2018
PubMed

Insights

High-throughput CRISPR-Cas9 screens reveal that specific guide RNA sequences can harm or kill E. coli. Tuning dCas9 expression and understanding off-target effects are key for safe CRISPR-Cas9 use in bacteria.

Area of Science:

  • Molecular Biology
  • Genetics
  • Microbiology

Background:

  • CRISPR-Cas9 screens are vital for understanding gene function and interactions.
  • Catalytically dead Cas9 (dCas9) can be directed to block gene transcription.

Purpose of the Study:

  • To investigate the impact of guide RNA sequences on E. coli fitness using dCas9.
  • To identify design rules for safe dCas9 application in bacterial systems.

Main Methods:

  • Genome-wide CRISPR-Cas9 screens in Escherichia coli.
  • Utilizing a machine-learning approach to analyze guide RNA sequences.
  • Assessing gene transcription blocking and fitness defects.

Main Results:

  • Specific 5-nucleotide seed sequences in guide RNAs cause significant fitness defects or lethality in E. coli.
  • These effects are concentration-dependent and can be modulated by dCas9 expression levels.
  • Off-target binding with only nine nucleotides of homology can strongly inhibit gene expression.

Conclusions:

  • CRISPR-Cas9 guide RNA design requires careful consideration of seed sequences and homology for safe bacterial applications.
  • Tuning dCas9 expression is crucial for mitigating unintended consequences.
  • This study provides essential design guidelines for effective and safe dCas9 use in E. coli.

More Related Videos

Overexpressing and Purifying a Toxic Nuclease from Escherichia coli08:13

Overexpressing and Purifying a Toxic Nuclease from Escherichia coli

Published on: August 29, 2025

787
High Throughput Screening of Fungal Endoglucanase Activity in Escherichia coli06:16

High Throughput Screening of Fungal Endoglucanase Activity in Escherichia coli

Published on: August 13, 2011

21.1K

Related Concept Videos

Cis-regulatory Sequences02:02

Cis-regulatory Sequences

Cis-regulatory sequences are short fragments of non-coding DNA that are present on the same chromosomes as the genes that they regulate. These fragments serve as binding sites for transcriptional regulators, proteins that are responsible for controlling gene transcription and differential gene expression across cell types in eukaryotes. Cis-regulatory sequences can be close to the gene of interest or thousands of bases away in the DNA sequence; however, those sequences that are further away are...
11.9K
Chemotaxis in E. coli01:27

Chemotaxis in E. coli

Chemotaxis in Escherichia coli is a sensory-driven motility mechanism that enables bacteria to navigate chemical gradients, moving toward beneficial environments while avoiding harmful conditions. This process relies on a signal transduction system integrating external chemical cues with flagellar motor control.Chemoreceptors and Signal DetectionE. coli detects chemical gradients through methyl-accepting chemotaxis proteins (MCPs), which are membrane-bound chemoreceptors that sense attractants...
972
Toxic Reactions: Overview01:26

Toxic Reactions: Overview

When toxic substances penetrate the human body, they disseminate to various tissues, undergoing metabolic changes. This process yields reactive metabolites that may covalently bind with specific target molecules, resulting in toxicity.
Toxicity falls into two primary categories: local and systemic.
Local toxicity appears at the exposure site, such as protein denaturation caused by caustic substances.
In contrast, systemic toxicity requires the toxic agent's absorption and distribution,...
2.1K
Stringent Response in E. coli01:23

Stringent Response in E. coli

Bacterial growth is closely tied to nutrient availability, with cells proliferating exponentially under favorable conditions and entering a stationary phase when resources become scarce. This transition is mediated by a regulatory mechanism known as the stringent response, which allows bacteria to adapt to nutrient deprivation by modulating gene expression and metabolic activity.During nutrient scarcity, intracellular amino acid levels decline. It results in the accumulation of uncharged tRNAs...
379
Cell Specific Gene Expression01:58

Cell Specific Gene Expression

Multicellular organisms contain a variety of structurally and functionally distinct cell types, but the DNA in all the cells originated from the same parent cells. The differences in the cells can be attributed to the differential gene expression. Liver cells, whose functions include detoxification of blood, production of bile to metabolize fats, and synthesis of proteins essential for metabolism, must express a specific set of genes to perform their functions. Gene expression also varies with...
16.6K
Sequences01:29

Sequences

Sequences are fundamental mathematical objects consisting of ordered lists of numbers that follow a specific rule or pattern. Sequences are critical in various mathematical concepts, including calculus, series, and number theory. They can model real-world phenomena such as population growth, financial investments, and physical processes like the diminishing height of a bouncing ball.Each number in a sequence is referred to as a term. Typically, the terms are denoted as a1, a2, a3,…, where...
285