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Related Concept Videos

CRISPR/Cas9 Genome Editing01:28

CRISPR/Cas9 Genome Editing

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The CRISPR-Cas system serves as a bacterial defense mechanism against invading genetic elements such as viruses and plasmids, forming the foundation for its adaptation as a powerful genome-editing tool. Originally discovered in prokaryotes, this system has been repurposed to revolutionize genetic engineering across a wide range of organisms, including plants, animals, and humans. The core component, Cas9, is an endonuclease derived from Streptococcus pyogenes, capable of introducing...
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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: Jul 15, 2025

CIRCLE-Seq for Interrogation of Off-Target Gene Editing
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An AsCas12f-based compact genome-editing tool derived by deep mutational scanning and structural analysis.

Tomohiro Hino1, Satoshi N Omura2, Ryoya Nakagawa2

  • 1Department of Cardiovascular Medicine, Graduate School of Medical Science, Kyoto Prefectural University of Medicine, Kyoto 602-8566, Japan.

Cell
|September 30, 2023
PubMed
Summary

Engineered compact Cas12f variants (enAsCas12f) show enhanced genome editing in human cells and mice. These variants are suitable for adeno-associated virus (AAV) delivery, offering a minimal platform for in vivo gene therapy.

Keywords:
CRISPR-Casanimal experimentscryo-EMdeep mutational scanninggene therapygenome editingiPS cells

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Area of Science:

  • Molecular Biology
  • Gene Editing Technologies
  • Biotechnology

Background:

  • SpCas9 and AsCas12a are established genome-editing tools but are limited by their large size for adeno-associated virus (AAV) vector delivery.
  • Compact CRISPR systems are needed for efficient in vivo gene therapy applications.
  • The type V-F Cas12f from Acidibacillus sulfuroxidans is a naturally small genome-editing enzyme.

Purpose of the Study:

  • To enhance the genome-editing activity of the compact Cas12f enzyme from Acidibacillus sulfuroxidans.
  • To develop novel Cas12f variants suitable for AAV-mediated delivery and in vivo gene editing.
  • To investigate the structural basis for enhanced Cas12f activity.

Main Methods:

  • Deep mutational scanning was employed to identify beneficial mutations in Cas12f.
  • Structure-informed design principles were used to rationally engineer Cas12f variants.
  • Cryo-electron microscopy (cryo-EM) was utilized to determine the structural basis of enhanced activity.

Main Results:

  • Two enhanced Cas12f variants (enAsCas12f) with significantly improved genome-editing activity in human cells were generated.
  • The enAsCas12f variants demonstrated comparable editing efficiency to SpCas9 and AsCas12a.
  • Cryo-EM structures revealed that mutations enhance dimer formation and nucleic acid interactions, leading to increased DNA cleavage.

Conclusions:

  • Enhanced Cas12f variants provide a compact and efficient genome-editing platform for in vivo applications.
  • The developed enAsCas12f system is compatible with all-in-one AAV vectors for gene therapy.
  • enAsCas12f exhibits efficient gene knock-in/knock-out and transcriptional activation in vivo, paving the way for minimal gene editing tools.