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Related Concept Videos

CRISPR/Cas9 Genome Editing01:28

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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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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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Updated: Mar 30, 2026

Enhanced Genome Editing with Cas9 Ribonucleoprotein in Diverse Cells and Organisms
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In Vitro CRISPR/Cas9 System for Efficient Targeted DNA Editing.

Yunkun Liu1, Weixin Tao1, Shishi Wen1

  • 1Key Laboratory of Combinatorial Biosynthesis and Drug Discovery, Ministry of Education, and Wuhan University School of Pharmaceutical Sciences, Wuhan, People's Republic of China.

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Summary

We developed an in vitro CRISPR editing (ICE) system for precise DNA manipulation. This tool efficiently refactors large DNA fragments, including biosynthetic gene clusters, enabling new avenues for natural product discovery and engineering.

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Area of Science:

  • Molecular Biology
  • Synthetic Biology
  • Genomics

Background:

  • The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) system is widely used for in vivo genome editing.
  • In vitro applications of CRISPR/Cas9 for large DNA fragment manipulation, particularly for refactoring biosynthetic gene clusters, remain less explored.

Purpose of the Study:

  • To establish a highly efficient in vitro CRISPR/Cas9-mediated editing (ICE) system for precise manipulation of large DNA fragments.
  • To demonstrate the system's capability in refactoring biosynthetic gene clusters in Streptomyces.
  • To improve the accuracy and efficiency of in vitro DNA editing.

Main Methods:

  • Investigated Cas9 cleavage of circular pUC18 DNA to understand error generation.
  • Utilized T4 DNA polymerase for end repair to enhance editing accuracy.
  • Applied the ICE system to delete genes within antibiotic biosynthetic gene clusters (RK-682 and holomycin) and insert a resistance gene.

Main Results:

  • Cas9 cleavage of DNA resulted in random nucleotide deletions at the editing site.
  • T4 DNA polymerase efficiently repaired Cas9-generated ends, significantly improving editing accuracy.
  • Successfully deleted target genes (rkD, homE) and inserted a gene (bla) into large DNA fragments containing biosynthetic gene clusters.

Conclusions:

  • The in vitro CRISPR editing (ICE) system is a rapid, seamless, and highly efficient tool for DNA fragment editing.
  • ICE provides a powerful new method for investigating and engineering complex biosynthetic gene clusters.
  • This system advances the manipulation of natural product pathways for potential biotechnological applications.