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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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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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Precise Base Substitution Using CRISPR/Cas-Mediated Base Editor in Rice.

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PubMed
Summary

This study introduces SpCas9-NG, an engineered base editor that expands genome editing capabilities. It utilizes a relaxed NG protospacer adjacent motif (PAM) to enable precise base conversions at more targetable sites without DNA breaks.

Keywords:
Adenine base editorsAgrobacterium-mediated transformationBase substitutionCRISPR/CasCytosine base editors

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Area of Science:

  • Molecular Biology
  • Genome Engineering
  • Biotechnology

Background:

  • CRISPR/Cas systems offer precise genome editing without double-strand breaks.
  • Protospacer adjacent motif (PAM) sequences limit the targetable range of base editors.
  • Standard SpCas9 requires an NGG PAM, restricting editing sites.

Purpose of the Study:

  • To describe a protocol for base editing using the engineered SpCas9-NG variant.
  • To expand the flexibility and targetable range of base editing applications.
  • To enable base substitutions at sites with relaxed NG PAM sequences.

Main Methods:

  • Utilizing the engineered SpCas9-NG variant for base editing.
  • Implementing a protocol for precise base conversion at target DNA sites.
  • Leveraging altered PAM recognition to increase editing accessibility.

Main Results:

  • SpCas9-NG recognizes relaxed NG PAM sequences.
  • Expanded targetable range for base editing applications.
  • Facilitated precise base conversion without inducing DNA double-strand breaks.

Conclusions:

  • The SpCas9-NG base editor enhances the versatility of CRISPR-based genome editing.
  • This protocol allows for broader application of base editing by overcoming PAM limitations.
  • Precise genome editing is made more accessible with relaxed PAM recognition.