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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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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 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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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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Related Experiment Video

Updated: Jun 26, 2025

Enhanced Genome Editing with Cas9 Ribonucleoprotein in Diverse Cells and Organisms
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Engineering miniature CRISPR-Cas Un1Cas12f1 for efficient base editing.

Yueer Hu1, Linxiao Han1, Qiqin Mo1

  • 1Gene Editing Center, School of Life Science and Technology, ShanghaiTech University, Shanghai, China.

Molecular Therapy. Nucleic Acids
|May 20, 2024
PubMed
Summary

New miniature base editors (STUminiBEs) overcome adeno-associated virus cargo limits for gene therapy. These hypercompact editors achieve high efficiency for A-to-G and C-to-T conversions, enabling potential biomedical applications.

Keywords:
AAVMT: RNA/DNA EditingSso7dUn1Cas12f1base editingminiature CRISPRprotein engineering

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Genome Editing in Mammalian Cell Lines using CRISPR-Cas
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Genome Editing in Mammalian Cell Lines using CRISPR-Cas

Published on: April 11, 2019

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Area of Science:

  • Molecular Biology
  • Gene Editing Technologies
  • Biomedical Engineering

Background:

  • Adeno-associated virus (AAV) is a promising gene therapy vector but has a limited cargo capacity (4.7 kb).
  • Existing SpCas9-mediated base editors exceed AAV packaging limits, hindering their clinical use.
  • The development of compact and efficient base editors is crucial for advancing in vivo gene therapy.

Purpose of the Study:

  • To engineer hypercompact and highly efficient base editors suitable for AAV packaging.
  • To enhance the base-editing efficiency of the Un1Cas12f1 system.
  • To demonstrate the therapeutic potential of these miniature base editors in mammalian cells.

Main Methods:

  • Engineering the Un1Cas12f1 protein and truncating single-guide RNA (sgRNA) to create miniature base editors (STUminiBEs).
  • Fusing the non-specific DNA binding protein Sso7d to enhance editor activity.
  • Packaging STUminiBEs into AAV vectors for delivery and testing in mammalian cells.

Main Results:

  • Achieved robust A-to-G base editing with STUminiABEs (54% average efficiency).
  • Demonstrated efficient C-to-T base editing with STUminiCBEs (45% average efficiency).
  • Successfully introduced a premature stop codon in the PCSK9 gene using AAV-packaged STUminiCBEs in mammalian cells.

Conclusions:

  • STUminiBEs represent a significant advancement in miniature base editor technology.
  • These editors are efficiently packaged into AAVs, overcoming previous size limitations.
  • STUminiBEs hold great promise for future biological research and biomedical applications in gene therapy.