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Related Concept Videos

CRISPR01:59

CRISPR

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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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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.
The donor site from where the transposon is excised is either degraded or...
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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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Homologous Recombination02:31

Homologous Recombination

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The basic reaction of homologous recombination (HR) involves two chromatids that contain DNA sequences sharing a significant stretch of identity. One of these sequences uses a strand from another as a template to synthesize DNA in an enzyme-catalyzed reaction. The final product is a novel amalgamation of the two substrates. To ensure an accurate recombination of sequences, HR is restricted to the S and G2 phases of the cell cycle. At these stages, the DNA has been replicated already and the...
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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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Experimental RNAi02:15

Experimental RNAi

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RNA interference (RNAi) is a cellular mechanism that inhibits gene expression by suppressing its transcription or activating the RNA degradation process. The mechanism was discovered by Andrew Fire and Craig Mello in 1998 in plants. Today, it is observed in almost all eukaryotes, including protozoa, flies, nematodes, insects, parasites, and mammals. This precise cellular mechanism of gene silencing has been developed into a technique that provides an efficient way to identify and determine the...
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Related Experiment Video

Updated: Jun 3, 2025

Enhanced Genome Editing with Cas9 Ribonucleoprotein in Diverse Cells and Organisms
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Enhanced Genome Editing with Cas9 Ribonucleoprotein in Diverse Cells and Organisms

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CRISPR-Enabled Autonomous Transposable Element (CREATE) for RNA-based gene editing and delivery.

Yuxiao Wang1, Ruei-Zeng Lin2, Meghan Harris2

  • 1Myeloid Therapeutics Inc., Cambridge, MA, 02139, USA. ywang@myeloidtx.com.

EMBO Reports
|January 9, 2025
PubMed
Summary

A new genome editing technology called CRISPR-Enabled Autonomous Transposable Element (CREATE) allows precise gene insertion without DNA breaks. This RNA-based system shows potential for treating genetic diseases by delivering large genes effectively.

Keywords:
CRISPR/Cas9Gene EditingGene TherapyRetrotransposon

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Selection-dependent and Independent Generation of CRISPR/Cas9-mediated Gene Knockouts in Mammalian Cells
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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
  • Gene Editing Technologies
  • Genetic Engineering

Background:

  • Existing genome editing tools often require double-strand breaks (DSBs) or DNA templates, limiting their application for large gene delivery.
  • There is a need for advanced genome editing systems capable of precise, template-free insertion of large genetic payloads.

Purpose of the Study:

  • To introduce and characterize CRISPR-Enabled Autonomous Transposable Element (CREATE), a novel RNA-based genome editing system.
  • To demonstrate the capability of CREATE for targeted insertion of large gene expression cassettes into specific genomic loci.

Main Methods:

  • Utilized a modified LINE-1 (L1) mRNA to encapsulate a payload gene for delivery.
  • Employed a CRISPR/Cas9 nickase to guide L1-mediated reverse transcription and integration at targeted genomic sites.
  • Validated CREATE system in human cell lines and primary T cells, assessing insertion specificity and off-target effects.

Main Results:

  • Successfully demonstrated programmable insertion of a 1.1 kb gene expression cassette into targeted genomic locations.
  • CREATE system operates without inducing double-strand breaks or requiring DNA templates for gene integration.
  • Mechanistic studies confirmed high editing specificity with no detectable off-target events.

Conclusions:

  • CREATE represents a programmable, RNA-based gene delivery technology with significant therapeutic potential for genetic diseases.
  • The system overcomes limitations of traditional genome editing by enabling template-free, precise insertion of large genes.