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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.
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Because the DNA segments are cut and reorganized in a direction-specific manner, site-specific recombination has emerged as an efficient genetic engineering technique. Flippase and Cyclization recombinases or Flp and Cre, respectively, are two members of the tyrosine recombinase family derived from bacteriophages, that are used to mediate site-specific DNA insertions, deletions, and targeted expression of proteins in mammalian cell lines.
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DNA-only Transposons02:57

DNA-only Transposons

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DNA-only transposons are called autonomous transposons since they code for the enzyme transposase that is required for the transposition mechanism. Insertion of transposons can alter gene functions in multiple ways. They can mutate the gene, alter gene expression by introducing a novel promoter or insulator sequence, introduce new splice sites, and change the mRNA transcripts produced, or remodel chromatin structure.
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Transposons01:24

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Transposons, or "jumping genes," are small mobile genetic elements (MGEs) that range from 700 to 40,000 base pairs in length. They are found in all organisms and can move within the same chromosome or transfer to different chromosomes. In some cases, transposons can also jump between different host DNA molecules, such as plasmids or viruses, contributing to genetic variability.Barbara McClintock first discovered these mobile genetic elements in the 1940s while studying maize genetics, and she...
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Related Experiment Video

Updated: Aug 5, 2025

Genome Editing in Mammalian Cell Lines using CRISPR-Cas
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Bacterial genome engineering using CRISPR RNA-guided transposases.

Diego R Gelsinger1, Phuc Leo H Vo2, Sanne E Klompe1

  • 1Department of Biochemistry and Molecular Biophysics, Columbia University, New York, NY, USA.

Biorxiv : the Preprint Server for Biology
|March 30, 2023
PubMed
Summary
This summary is machine-generated.

CRISPR-associated transposons (CASTs) enable efficient, kilobase-scale bacterial genome engineering without homologous recombination. This protocol details using CAST systems for precise DNA integration in diverse species within one week.

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Area of Science:

  • Microbiology
  • Molecular Biology
  • Synthetic Biology

Background:

  • CRISPR-associated transposons (CASTs) offer a novel platform for large-scale genome engineering.
  • Traditional methods often require homologous recombination and can be inefficient for substantial DNA insertions.

Approach:

  • This study presents a detailed protocol for utilizing CAST systems in bacterial genome engineering.
  • The protocol covers guide RNA and DNA payload customization, delivery methods, and analysis of integration events.
  • A computational algorithm for crRNA design and a pipeline for multiplexed DNA insertion are also described.

Key Points:

  • CAST systems facilitate accurate, programmable integration of large genetic payloads (~kilobases) in diverse Gram-negative bacteria.
  • CRISPR RNA-guided transposases achieve near 100% insertion efficiency in *E. coli*.
  • Multiplexed edits and targeted insertions are achievable using multiple guides and specialized pipelines.

Conclusions:

  • CAST systems represent a transformative technology for kilobase-scale genome engineering.
  • The provided protocol enables rapid isolation of engineered bacterial strains using standard molecular biology techniques.
  • This approach simplifies and enhances the efficiency of creating complex genomic modifications.