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Chemical modifications enable versatile Cas9 protein engineering for enhanced gene editing. This platform facilitates precise DNA repair and novel therapeutic applications, including engineered cell lines for disease treatment.

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Area of Science:

  • Biochemistry
  • Molecular Biology
  • Gene Editing Technologies

Background:

  • Current Cas9 gene editing relies on genetic modifications at protein termini, limiting functional diversity.
  • A need exists for versatile Cas9 engineering to incorporate a wider range of molecules for enhanced gene editing capabilities.

Purpose of the Study:

  • To develop a chemical modification platform for site-specific and multi-site conjugation of diverse molecules to Cas9.
  • To enhance precision genome editing efficiency by optimizing single-stranded oligonucleotide donor (ssODN) incorporation.
  • To engineer cell lines for therapeutic protein secretion using the developed Cas9 conjugation platform.

Main Methods:

  • Chemical conjugation techniques for site-specific and multiple-site modification of Cas9 protein.
  • Integration of single-stranded oligonucleotide donors (ssODNs) at DNA break sites via modified Cas9.
  • Engineering of INS-1E beta-cell line for glucose-dependent interleukin-10 secretion.

Main Results:

  • Successful development of a platform for chemical modification of Cas9 at internal and terminal sites.
  • Demonstrated significant increase in precision genome editing efficiency through multiple-site ssODN conjugation to Cas9.
  • Engineered INS-1E cells to secrete interleukin-10 in a glucose-dependent manner, repurposing insulin secretion machinery.

Conclusions:

  • Chemical modification of Cas9 provides a versatile platform for diverse functionalization, overcoming limitations of genetic fusion.
  • This platform enhances precision genome editing efficiency and is compatible with various ssODN lengths.
  • The developed conjugation strategy enables novel therapeutic applications, such as engineering cells for controlled protein secretion.