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In-vitro Mutagenesis01:16

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To learn more about the function of a gene, researchers can observe what happens when the gene is inactivated or “knocked out,” by creating genetically engineered knockout animals. Knockout mice have been particularly useful as models for human diseases such as cancer, Parkinson’s disease, and diabetes.
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Related Experiment Video

Updated: Jun 3, 2025

Efficient Gene Knockdown in the Liver via Intrasplenic Injection of Adeno-Associated Virus Serotype 8 (AAV8)-Delivered Small Hairpin RNA
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Efficient Gene Knockdown in the Liver via Intrasplenic Injection of Adeno-Associated Virus Serotype 8 (AAV8)-Delivered Small Hairpin RNA

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KOnezumi-AID: Automation Software for Efficient Multiplex Gene Knockout Using Target-AID.

Taito Taki1, Kento Morimoto2, Seiya Mizuno3

  • 1College of Biological Sciences, University of Tsukuba, Tsukuba 305-8572, Japan.

International Journal of Molecular Sciences
|January 8, 2025
PubMed
Summary

KOnezumi-AID is a new tool for designing guide RNAs (gRNAs) for Target-AID gene knockout. It enables efficient, simultaneous multi-gene editing without DNA breaks, advancing genome editing research.

Keywords:
CRISPRTarget-AIDbase editingbioinformaticsgene knockoutgenome editingmultiple base editingmultiple gene knockoutsoftware

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Area of Science:

  • Genomics
  • Molecular Biology
  • Bioinformatics

Background:

  • CRISPR-Cas9 enables efficient gene knockout but risks chromosomal rearrangements, hindering simultaneous multi-gene editing.
  • Base editors like Target-AID offer precise modifications without DNA double-strand breaks, addressing safety concerns.
  • Existing tools lack support for Target-AID-based simultaneous multi-gene knockout design.

Purpose of the Study:

  • To develop KOnezumi-AID, a command-line tool for designing guide RNAs (gRNAs) for Target-AID-mediated genome editing.
  • To facilitate simultaneous gene knockout by inducing premature termination codons or exon skipping.
  • To provide experiment-ready gRNA designs for mouse and human genomes.

Main Methods:

  • Developed KOnezumi-AID, a command-line tool for gRNA design.
  • Implemented algorithms to induce premature termination codons or promote exon skipping for gene knockout.
  • Enabled batch processing for large-scale genome editing and CRISPR screening.

Main Results:

  • KOnezumi-AID generates experiment-ready gRNA designs for Target-AID-mediated gene knockout.
  • The tool supports simultaneous knockout of multiple genes in mouse and human genomes.
  • Batch processing allows rapid and precise gRNA design for large-scale applications.

Conclusions:

  • KOnezumi-AID is an efficient and user-friendly tool for gRNA design in Target-AID genome editing.
  • It streamlines genome editing workflows, particularly for simultaneous multi-gene knockout.
  • The tool advances gene knockout research by providing a comprehensive solution for gRNA design.