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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.
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CRISPR stands for Clustered Regularly Interspaced Short Palindromic Repeats is a adaptive immune system found in bacteria and archaea that protects against viral infections. This system enables prokaryotic cells to identify, remember, and neutralize foreign genetic elements, primarily bacteriophages, by storing fragments of the invader’s DNA as a genetic memory.The CRISPR immune response begins during an initial infection. Cas (CRISPR-associated) proteins play a central role in this...
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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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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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The organization of prokaryotic genes in their genome is notably different from that of eukaryotes. Prokaryotic genes are organized, such that the genes for proteins involved in the same biochemical process or function are located together in groups. This group of genes, along with their regulatory elements, are collectively known as an operon. The functional genes in an operon are transcribed together to give a single strand of mRNA known as polycistronic mRNA.
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Updated: Mar 7, 2026

Application of CRISPR Interference CRISPRi for Gene Silencing in Pathogenic Species of Leptospira
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Programmable transcriptional repression in mycobacteria using an orthogonal CRISPR interference platform.

Jeremy M Rock1, Forrest F Hopkins1, Alejandro Chavez2,3,4

  • 1Department of Immunology and Infectious Diseases, Harvard T.H. Chan School of Public Health, Boston, Massachusetts 02115, USA.

Nature Microbiology
|February 7, 2017
PubMed
Summary

A new CRISPR interference (CRISPRi) system using Streptococcus thermophilus Cas9 (dCas9Sth1) enables efficient gene silencing in Mycobacterium tuberculosis. This advance facilitates tuberculosis research, including drug synergy studies.

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Area of Science:

  • Microbiology and Molecular Biology
  • Genetics and Genomics
  • Drug Discovery and Development

Background:

  • Genetic manipulation in Mycobacterium tuberculosis is challenging, hindering the development of new tuberculosis (TB) treatments.
  • Existing CRISPR interference (CRISPRi) systems in M. tuberculosis exhibit limitations in gene knockdown efficiency and cause proteotoxicity.

Purpose of the Study:

  • To develop a more effective and scalable CRISPRi system for gene silencing in Mycobacterium tuberculosis.
  • To overcome the limitations of current CRISPRi systems, specifically poor knockdown efficiency and proteotoxicity.

Main Methods:

  • Screened eleven different Cas9 orthologues for functional gene knockdown in mycobacteria.
  • Identified and characterized the Streptococcus thermophilus Cas9 (dCas9Sth1) for its efficacy and robustness in gene silencing.
  • Utilized the dCas9Sth1 CRISPRi system to investigate gene function within bacterial operons and study drug synergies in the folate biosynthesis pathway.

Main Results:

  • Identified four functional Cas9 orthologues, with dCas9Sth1 demonstrating superior performance.
  • dCas9Sth1 achieved 20- to 100-fold knockdown of endogenous gene expression with minimal proteotoxicity.
  • dCas9Sth1-mediated knockdown was effective even when targeting sites distant from the transcriptional start site, enabling precise gene function analysis.

Conclusions:

  • The dCas9Sth1 CRISPRi system offers a robust, efficient, and scalable platform for gene silencing in Mycobacterium tuberculosis.
  • This system significantly improves upon existing CRISPRi tools, addressing limitations of efficiency and proteotoxicity.
  • The dCas9Sth1 CRISPRi system is expected to be broadly applicable for functional genomics, genetic interaction mapping, and drug-target profiling in TB research.