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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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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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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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Gene Digital Circuits Based on CRISPR-Cas Systems and Anti-CRISPR Proteins
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A Simplified Method for CRISPR-Cas9 Engineering of Bacillus subtilis.

Ankita J Sachla1, Alexander J Alfonso1, John D Helmann1

  • 1Department of Microbiology, Cornell Universitygrid.5386.8, Ithaca, New York, USA.

Microbiology Spectrum
|September 15, 2021
PubMed
Summary

This study introduces a simplified CRISPR-Cas9 genome editing method for Bacillus subtilis, eliminating guide RNA design and cloning. This approach accelerates bacterial strain construction and genetic manipulation using existing gene replacement libraries.

Keywords:
Bacillus subtilisCRISPR-Cas9allelic replacementgeneticsgenome editingtransformation

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Area of Science:

  • Microbiology and Molecular Biology
  • Bacterial Genetics and Genomics
  • Biotechnology and Synthetic Biology

Background:

  • The clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 system is a powerful tool for bacterial strain construction.
  • Conventional CRISPR-Cas9 editing in Bacillus subtilis typically requires designing and cloning specific guide RNAs (gRNAs) and repair templates for each genetic modification.
  • This process can be time-consuming and labor-intensive, hindering rapid genetic manipulation.

Purpose of the Study:

  • To develop a streamlined CRISPR-Cas9 genome editing strategy for Bacillus subtilis that bypasses the need for gRNA design and cloning.
  • To leverage the existing Bacillus subtilis Gene Engineering (BKE) collection of strains, each containing an integrated erythromycin resistance cassette.
  • To facilitate rapid and versatile genome modifications, including gene replacements, site-specific mutations, and reporter gene fusions.

Main Methods:

  • Utilized a single plasmid (pAJS23) encoding a gRNA targeting the erythromycin (erm) resistance cassette.
  • Co-transformed the plasmid with repair templates, which could be PCR products or genomic DNA, or engineered directly into the plasmid.
  • Demonstrated genome cleavage at nonessential genes and sites near essential genes within the BKE collection.

Main Results:

  • Successfully generated gene replacements, site-specific mutations, modifications of intergenic regions, and introduction of gene-reporter fusions.
  • The method effectively bypassed the requirement for designing and cloning individual gRNAs for each target gene.
  • Facilitated the facile transfer of mutations and genetic constructions without intermediate cloning steps.

Conclusions:

  • The presented strategy significantly simplifies CRISPR-Cas9-mediated genome editing in Bacillus subtilis.
  • This rapid method enables efficient genome manipulation across a large collection of pre-existing gene disruptants.
  • The approach accelerates the construction of bacterial strains and expands the utility of Bacillus subtilis as a model organism and biotechnological platform.