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Related Concept Videos

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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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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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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Within a biological system, the DNA encodes the RNA, and the nucleotide sequence in the RNA further defines the amino acid sequence in the protein. This is referred to as “The Central Dogma of Molecular Biology” - a term coined by Francis Crick.  Central dogma is a firm principle in biology that defines the flow of genetic information within any life form. The two fundamental steps in central dogma are - transcription and translation.
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Proteins are involved in several cellular processes and biochemical reactions. Analyzing a specific protein of interest requires it to be isolated from the other proteins in the cell. This is achieved by overexpressing the specific gene in a suitable host to produce large quantities of the target protein. A tag or label is recombined with the gene to produce a fusion protein containing the target protein and the tag. The tags on these fusion proteins can then be used for easy detection and...
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The structure and stability of mRNA molecules regulates gene expression, as mRNAs are a key step in the pathway from gene to protein. In eukaryotes, the half-life of mRNA varies from a few minutes up to several days. mRNA stability is essential in growth and development. The absence of the proteins regulating its stability, such as tristetraprolin in mice, can cause systemic issues, including bone marrow overgrowth, inflammation, and autoimmunity.
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Related Experiment Video

Updated: Jan 26, 2026

Efficient Production and Identification of CRISPR/Cas9-generated Gene Knockouts in the Model System Danio rerio
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LeishGEdit: A Method for Rapid Gene Knockout and Tagging Using CRISPR-Cas9.

Tom Beneke1, Eva Gluenz2

  • 1Sir William Dunn School of Pathology, University of Oxford, Oxford, UK.

Methods in Molecular Biology (Clifton, N.J.)
|April 14, 2019
PubMed
Summary

This study introduces a fast and scalable CRISPR-Cas9 method for gene editing in Leishmania. The new technique enables rapid gene knockout and protein tagging, accelerating Leishmania research.

Keywords:
CRISPRCas9Gene editingKinetoplastidsKnockoutLeishGEditLeishmaniaT7 RNA polymeraseTagging

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Selection-dependent and Independent Generation of CRISPR/Cas9-mediated Gene Knockouts in Mammalian Cells

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Area of Science:

  • Molecular Biology
  • Parasitology
  • Genetics

Background:

  • Postgenomic analysis of Leishmania requires efficient gene manipulation techniques.
  • Traditional homologous recombination methods for gene knockout/tagging in Leishmania are slow and labor-intensive, especially for multiple alleles.
  • CRISPR-Cas9 systems offer a promising alternative to overcome these limitations.

Purpose of the Study:

  • To detail a simple, rapid, and scalable method for CRISPR-Cas9-mediated gene knockout and tagging in Leishmania.
  • To provide a streamlined workflow for generating gene-edited Leishmania cell lines within a week.

Main Methods:

  • Utilized simple PCR to generate single-guide RNA (sgRNA) transcription templates and drug-selectable editing cassettes.
  • Employed a modular set of pT plasmids for knockout drug resistance gene amplification and pPLOT plasmids for N- or C-terminal protein tagging.
  • Integrated an online platform (LeishGEdit.net) for automated primer design, facilitating small-batch or 96-well plate transfections for large-scale screening.

Main Results:

  • Successfully developed and validated a CRISPR-Cas9-based system for efficient gene knockout and tagging in Leishmania.
  • Demonstrated the generation of knockout mutants or tagged cell lines within a one-week timeframe.
  • The method is scalable for high-throughput screening applications.

Conclusions:

  • The described CRISPR-Cas9 method significantly enhances the speed and efficiency of gene manipulation in Leishmania.
  • This approach facilitates rapid postgenomic analyses and accelerates the study of Leishmania biology.
  • The protocol is adaptable for both small-scale research and large-scale genetic screening.