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Related Concept Videos

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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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 1, 2025

CRISPR/Cas12a Multiplex Genome Editing of Saccharomyces cerevisiae and the Creation of Yeast Pixel Art
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Single-Nucleotide Microbial Genome Editing Using CRISPR-Cas12a.

Ho Joung Lee1, Sang Jun Lee2

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

Methods in Molecular Biology (Clifton, N.J.)
|March 12, 2024
PubMed
Summary
This summary is machine-generated.

Researchers achieved precise single-nucleotide genome editing in E. coli using a modified CRISPR-Cas12a system and a mutagenic DNA donor. This method allows for exact genetic modifications in microbial cells.

Keywords:
3′-truncated crRNAPrecise genome editingSingle-baseCas12a

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Area of Science:

  • Microbiology
  • Molecular Biology
  • Genetics

Background:

  • Microbial genome editing is crucial for synthetic biology and biotechnology.
  • Existing methods like donor DNA-directed mutagenesis and CRISPR-Cas12a have limitations in precision.
  • Single-nucleotide resolution in microbial editing is highly desirable for precise genetic manipulation.

Purpose of the Study:

  • To develop a method for single-nucleotide-level genome editing in E. coli.
  • To investigate the role of crRNA truncation in CRISPR-Cas12a mediated editing.
  • To achieve precise substitutions and indels at specific genomic loci.

Main Methods:

  • Utilized a mutagenic DNA oligonucleotide donor for precise editing.
  • Employed a CRISPR-Cas12a system with truncated crRNAs.
  • Targeted the galK gene in the E. coli genome for editing.

Main Results:

  • Successfully achieved single-nucleotide substitutions and indels in the E. coli genome.
  • Demonstrated that maximal truncation of the crRNA 3'-end enhances Cas12a-mediated editing precision.
  • Showcased precise editing at the galK locus.

Conclusions:

  • Maximal crRNA truncation is key for high-fidelity, single-nucleotide editing with CRISPR-Cas12a in E. coli.
  • This approach enables precise genetic engineering of microbial genomes.
  • The developed method offers a powerful tool for microbial strain development and functional genomics.