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RNA-seq03:21

RNA-seq

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RNA sequencing, or RNA-Seq, is a high-throughput sequencing technology used to study the transcriptome of a cell. Transcriptomics helps to interpret the functional elements of a genome and identify the molecular constituents of an organism. Additionally, it also helps in understanding the development of an organism and the occurrence of diseases. 
Before the discovery of RNA-seq, microarray-based methods and Sanger sequencing were used for transcriptome analysis. However, while...
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CRISPR-based Shuttle Cloning: A High-throughput Cloning Method
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High-throughput retrieval of target sequences from complex clone libraries using CRISPRi.

Ján Burian1, Vincent K Libis1, Yozen A Hernandez1

  • 1Laboratory of Genetically Encoded Small Molecules, Rockefeller University, New York, NY, USA.

Nature Biotechnology
|November 21, 2022
PubMed
Summary
This summary is machine-generated.

This study introduces a CRISPR counter-selection interruption circuit (CCIC) to retrieve specific microbial DNA clones from large libraries. This method enables faster access to genetic diversity previously unreachable by traditional culture-dependent techniques.

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Area of Science:

  • Microbiology
  • Genomics
  • Molecular Biology

Background:

  • Culture-dependent methods limit the study of microbial diversity.
  • Metagenomic DNA capture in clone libraries offers access to uncultured microbes.
  • Efficient retrieval of target clones from complex libraries is challenging.

Purpose of the Study:

  • To develop a novel method for retrieving specific clones from metagenomic libraries.
  • To enable the study of microbial genetic diversity inaccessible by traditional methods.
  • To combine CRISPR technology with cloning for enhanced genetic exploration.

Main Methods:

  • Utilized nuclease-deficient Cas9 to establish a CRISPR counter-selection interruption circuit (CCIC).
  • Applied CCIC for targeted clone retrieval from complex metagenomic libraries.
  • Integrated CCIC cloning with modern sequencing techniques.

Main Results:

  • Successfully established a functional CCIC for clone selection.
  • Demonstrated the ability of CCIC to retrieve target clones from complex libraries.
  • Facilitated rapid physical access to microbial genetic diversity.

Conclusions:

  • CRISPR counter-selection interruption circuit (CCIC) is an effective tool for retrieving target clones.
  • The CCIC method significantly enhances the study of microbial diversity in natural ecosystems.
  • Combining CCIC with sequencing provides rapid access to unexplored genetic resources.