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Related Concept Videos

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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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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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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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CIRCLE-Seq for Interrogation of Off-Target Gene Editing
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Engineered IscB-ωRNA system with expanded target range for base editing.

Qingquan Xiao1,2, Guoling Li1, Dingyi Han2,3

  • 1HuidaGene Therapeutics Co. Ltd., Shanghai, China.

Nature Chemical Biology
|August 15, 2024
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Summary

Researchers identified novel IscB proteins for base editing, enhancing one to expand its targeting scope. These engineered IscB base editors show promise for research and treating genetic diseases like Duchenne muscular dystrophy.

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Area of Science:

  • Molecular Biology
  • Gene Editing Technologies
  • Microbial Genomics

Background:

  • IscB proteins are evolutionary ancestors of Cas9 nucleases, functioning as RNA-guided DNA endonucleases and nickases.
  • Their compact nature makes them suitable for base editing applications, but limited targeting scope restricts their utility.
  • Identifying IscB proteins with diverse target-adjacent motif (TAM) recognition is crucial for expanding base editing capabilities.

Purpose of the Study:

  • To discover novel IscB proteins with activity in mammalian cells.
  • To engineer existing IscB proteins to enhance activity and broaden TAM recognition.
  • To develop IscB-derived base editors for therapeutic applications, including genetic disease correction.

Main Methods:

  • Screening of uncharacterized IscB proteins from uncultured microbes.
  • Protein and ωRNA engineering to enhance IscB activity and alter TAM specificity.
  • Fusion of deaminase domains with engineered IscB nickases to create base editors.
  • In vitro and in vivo validation of base editor efficiency in mammalian cells and disease models.

Main Results:

  • Identified 10 active IscB proteins from 19 uncharacterized microbial candidates.
  • Engineered IscB.m16 to create IscB.m16*, expanding TAM scope from MRNRAA to NNNGNA.
  • Developed IscB.m16*-derived base editors demonstrating robust efficiency in mammalian cells.
  • Successfully restored Duchenne muscular dystrophy proteins in mice using single AAV delivery of base editors.

Conclusions:

  • This study provides a suite of compact base-editing tools derived from novel and engineered IscB proteins.
  • The expanded TAM scope of IscB.m16* enhances its versatility for gene editing.
  • IscB-based base editors hold significant potential for both fundamental research and therapeutic interventions in genetic disorders.