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Related Concept Videos

CRISPR/Cas9 Genome Editing01:28

CRISPR/Cas9 Genome Editing

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The CRISPR-Cas system serves as a bacterial defense mechanism against invading genetic elements such as viruses and plasmids, forming the foundation for its adaptation as a powerful genome-editing tool. Originally discovered in prokaryotes, this system has been repurposed to revolutionize genetic engineering across a wide range of organisms, including plants, animals, and humans. The core component, Cas9, is an endonuclease derived from Streptococcus pyogenes, capable of introducing...
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CRISPR and crRNAs02:53

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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.
The CRISPR-Cas system stores a copy of foreign DNA in the host genome and uses it to identify the foreign DNA upon reinfection. CRISPR-Cas has three different...
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CRISPR01:59

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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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Related Experiment Video

Updated: Oct 15, 2025

Genome-Wide CRISPR Screen for Unveiling Radiosensitive and Radioresistant Genes
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Genome-Wide CRISPR Screen for Unveiling Radiosensitive and Radioresistant Genes

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New Method for Genome-Scale Functional Genomic Study in Bacteria with Superior Performance: CRISPR Interference

Xihao Liao1, Xin-Hui Xing1,2, Chong Zhang3,4

  • 1MOE Key Laboratory for Industrial Biocatalysis, Institute of Biochemical Engineering, Department of Chemical Engineering, Tsinghua University, Beijing, China.

Methods in Molecular Biology (Clifton, N.J.)
|October 28, 2021
PubMed
Summary

We present a detailed protocol for pooled CRISPR screens in Escherichia coli to identify gene function and genotype-phenotype links. This method enhances genome-wide genetic screening capabilities for bacterial systems.

Keywords:
CRISPRiComputational biologyHigh-throughput screenMicrobial functional genomicsNext-generation sequencing

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Area of Science:

  • Molecular Biology
  • Genetics
  • Microbiology

Background:

  • High-throughput genetic screens are crucial for understanding gene function and genotype-phenotype associations.
  • CRISPR/Cas9 technology offers a powerful approach for genome-wide genetic analysis.
  • Efficient methods are needed for applying these screens in bacterial systems like Escherichia coli.

Purpose of the Study:

  • To provide a detailed protocol for conducting and evaluating pooled CRISPR screens in Escherichia coli.
  • To enable genome-wide identification of gene function and genotype-phenotype associations using CRISPR technology.
  • To offer a robust workflow adaptable for other bacterial systems.

Main Methods:

  • Guide RNA library design and construction for CRISPR interference.
  • Genome-scale pooled CRISPR screening in Escherichia coli.
  • Next-generation sequencing data processing and analysis.
  • Comparison with existing methods like transposon sequencing (Tn-seq).

Main Results:

  • The developed protocol facilitates pooled CRISPR screens in Escherichia coli.
  • The workflow allows for genome-wide identification of gene function.
  • CRISPR screens demonstrated superior performance compared to Tn-seq for similar library sizes and short gene lengths.
  • The protocol is adaptable for implementation in other bacterial systems.

Conclusions:

  • Pooled CRISPR screens provide a powerful and efficient tool for bacterial genetics.
  • The detailed protocol enables robust genome-wide functional genomics in Escherichia coli.
  • This methodology advances the study of genotype-phenotype relationships in diverse bacterial species.