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CRISPR01:59

CRISPR

50.9K
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

CRISPR and crRNAs

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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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Pre-mRNA Processing: RNA Splicing01:36

Pre-mRNA Processing: RNA Splicing

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Conservative Site-specific Recombination and Phase Variation02:53

Conservative Site-specific Recombination and Phase Variation

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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.
The recognition sites for Cre recombinase called LoxP...
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RNA Splicing01:32

RNA Splicing

56.3K
Splicing is the process by which eukaryotic RNA is edited before its translation into protein. The RNA strand transcribed from eukaryotic DNA is called the primary transcript. The primary transcripts that become mRNAs are called precursor messenger RNAs (pre-mRNAs). Eukaryotic pre-mRNA contains alternating sequences of exons and introns. Exons are nucleotide sequences that code for proteins, whereas introns are the non-coding regions. In RNA splicing, introns are removed and exons are bonded...
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Alternative RNA Splicing02:18

Alternative RNA Splicing

21.1K
Alternative RNA splicing is the regulated splicing of exons and introns to produce different mature mRNAs from a single pre-mRNA. Unlike in constitutive splicing where a single gene produces a single type of mRNA, alternative splicing allows an organism to produce multiple proteins from a single gene and plays an important role in protein diversity.
There are five types of alternative RNA splicing that vary in the ways the pre-mRNA segments are removed or retained in the mature mRNA. The first...
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Related Experiment Video

Updated: Jun 30, 2025

Designing, Packaging, and Delivery of High Titer CRISPR Retro and Lentiviruses via Stereotaxic Injection
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Designing, Packaging, and Delivery of High Titer CRISPR Retro and Lentiviruses via Stereotaxic Injection

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Repurposing CRISPR-Cas13 systems for robust mRNA trans-splicing.

David N Fiflis1, Nicolas A Rey2, Harshitha Venugopal-Lavanya1

  • 1Department of Biomedical Engineering, Duke University, Durham, NC, USA.

Nature Communications
|March 15, 2024
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Summary

CRISPR-Cas13 systems enable CRISPR Assisted mRNA Fragment Trans-splicing (CRAFT) for large mRNA editing. This novel RNA editing tool facilitates trans-splicing of RNA fragments into endogenous transcripts.

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Area of Science:

  • Molecular Biology
  • Gene Editing
  • RNA Biology

Background:

  • Type VI CRISPR enzymes, particularly Cas13, are established tools for RNA editing, including single base edits, demethylation, and RNA cleavage.
  • Current RNA editing methods face limitations in modifying extensive segments of messenger RNA (mRNA) transcripts.

Purpose of the Study:

  • To develop and validate a novel RNA editing technique, CRISPR Assisted mRNA Fragment Trans-splicing (CRAFT), utilizing CRISPR-Cas13 systems.
  • To enable the editing of large portions of endogenous mRNA transcripts by facilitating the trans-splicing of exogenous RNA fragments.

Main Methods:

  • Repurposing CRISPR-Cas13 systems for RNA trans-splicing.
  • Employing split reporter assays to evaluate orthogonal Cas13 systems.
  • Optimizing guide RNA length and identifying optimal trans-splicing sites within intronic regions.

Main Results:

  • Demonstrated successful trans-splicing of exogenous RNA fragments into endogenous pre-mRNA transcripts using the CRAFT method.
  • Achieved significantly enhanced editing of large 5' and 3' mRNA segments across diverse mammalian cell types.
  • Outperformed existing spliceosome-mediated trans-splicing methods in terms of editing efficiency for large segments.

Conclusions:

  • CRAFT represents a versatile platform for substantial mRNA transcript modification.
  • Applications include attaching protein tags, studying multiple mutations or SNPs, modifying untranslated regions (UTRs), and replacing large mRNA segments.