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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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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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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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Related Experiment Video

Updated: Jul 25, 2025

Enhanced Genome Editing with Cas9 Ribonucleoprotein in Diverse Cells and Organisms
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Multiplex Single-Nucleotide Microbial Genome Editing Achieved by CRISPR-Cas9 Using 5'-End-Truncated sgRNAs.

Se Ra Lim1, Ho Joung Lee1, Hyun Ju Kim1

  • 1Department of Systems Biotechnology and Institute of Microbiomics, Chung-Ang University, Anseong 17546, Republic of Korea.

ACS Synthetic Biology
|June 27, 2023
PubMed
Summary

This study introduces a novel truncated single-molecular guide RNA (sgRNA) method for highly accurate multiplex genome editing in Escherichia coli. This approach enables simultaneous, single-nucleotide edits in multiple genes, advancing synthetic biology applications.

Keywords:
CRISPR-Casmultiplexsingle-nucleotide editingtruncated sgRNA

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Area of Science:

  • Molecular Biology
  • Genetics
  • Synthetic Biology

Background:

  • Multiplex genome editing using CRISPR-Cas9 is valuable for efficiency but faces accuracy challenges.
  • Simultaneous editing of multiple genes requires precise control over guide RNA function.

Purpose of the Study:

  • To develop a highly accurate method for multiplex genome editing in Escherichia coli.
  • To demonstrate the efficacy of truncated single-molecular guide RNAs (sgRNAs) for simultaneous gene editing.

Main Methods:

  • Utilized a 5'-end-truncated single-molecular guide RNA (sgRNA) strategy for CRISPR-Cas9 mediated genome editing.
  • Applied the method to simultaneously edit two and three genes (galK, xylB, srlD) in Escherichia coli at single-nucleotide resolution.
  • Tested the method on targeted editing of cI and ilvG genes in E. coli.

Main Results:

  • Achieved highly efficient, single-nucleotide level simultaneous editing of galK and xylB genes.
  • Successfully demonstrated simultaneous editing of three genes (galK, xylB, and srlD) with single-nucleotide resolution.
  • Truncated sgRNAs enabled 30% efficiency in simultaneous editing of cI and ilvG genes, unlike untruncated sgRNAs.

Conclusions:

  • The truncated sgRNA method significantly enhances accuracy and efficiency in multiplex genome editing.
  • This technique allows for precise genetic modifications in Escherichia coli, with practical implications for synthetic biology.
  • The developed method shows potential for broad application in creating engineered organisms.