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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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CRISPR/Cas9-mediated Targeted Integration In Vivo Using a Homology-mediated End Joining-based Strategy
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CRISPR/Cas-Mediated In Planta Gene Targeting.

Simon Schiml1, Friedrich Fauser1,2, Holger Puchta3

  • 1Botanical Institute II, Karlsruhe Institute of Technology, 6980, 76049, Karlsruhe, Germany.

Methods in Molecular Biology (Clifton, N.J.)
|April 26, 2017
PubMed
Summary

We developed a new CRISPR/Cas9 gene targeting system for plants that bypasses the need for high transformation efficiencies. This in planta gene targeting (ipGT) system enables precise genome engineering by activating both the target and the donor DNA simultaneously.

Keywords:
Cas9Double-strand break repairEngineered nucleasesGene technologyGenome engineering

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Area of Science:

  • Plant biotechnology
  • Genome engineering
  • Molecular biology

Background:

  • CRISPR/Cas systems offer precise genome engineering across life forms.
  • In plants, targeted mutagenesis via Cas9-induced DNA double-strand breaks (DSBs) is common, repaired by nonhomologous end joining (NHEJ).
  • Site-specific alterations or transgene integration require homologous recombination (HR), which is challenging in plants due to prevalent NHEJ repair in somatic tissues.

Purpose of the Study:

  • To develop a novel in planta gene targeting (ipGT) system for plants.
  • To overcome the limitations of homologous recombination repair in plant somatic cells.
  • To enable precise genome engineering independent of high transformation efficiencies.

Main Methods:

  • Development of the in planta gene targeting (ipGT) system.
  • Utilizing Cas9 to induce DNA double-strand breaks (DSBs) at the target locus.
  • Simultaneous activation of the target site and the targeting vector.

Main Results:

  • The ipGT system activates both the target and the targeting vector concurrently.
  • This simultaneous activation facilitates precise gene targeting.
  • The system is independent of high plant transformation efficiencies.

Conclusions:

  • The novel ipGT system enhances the precision of plant genome engineering.
  • It provides a valuable tool for site-specific DNA alterations and transgene integration in plants.
  • This method simplifies gene targeting in plants by reducing reliance on transformation efficiency.