Engineering a CRISPR-associated IscB system for developing miniature genome-editing tools in human cells and mouse...

Area of Science:

• Molecular Biology
• Gene Editing Technologies
• Biotechnology

Background:

• IscB proteins are compact CRISPR-associated enzymes, potential alternatives to Cas9 for in vivo delivery.
• Existing IscB systems like OgeuIscB function in eukaryotes but require specific target-adjacent motifs (TAMs).
• There is a need for more efficient and versatile miniature genome editing tools.

Purpose of the Study:

• To characterize a novel CRISPR-associated IscB system, DelIscB, that recognizes a flexible TAM.
• To engineer DelIscB for enhanced activity and develop miniature base editors.
• To evaluate the in vivo applicability of engineered DelIscB systems.

Main Methods:

• Systematic engineering of DelIscB protein and its single-guide RNA (sgRNA).
• Fusion of engineered enDelIscB with T5 exonuclease (T5E) for DNA cleavage.
• Fusion of enDelIscB nickase with cytosine or adenosine deaminase for base editing.
• Generation of mouse models via microinjection of mRNA/sgRNA.

Main Results:

• Engineered enDelIscB showed a 48.9-fold increase in activity compared to the wild type.
• enDelIscB-T5E demonstrated efficient DNA cleavage in human cells, comparable to enIscB-T5E.
• Efficient miniature base editors (ICBE and IABE) were established using enDelIscB nickase.
• Successful generation of mouse models using enDelIscB and enDelIscB-T5E mRNA/sgRNA.

Conclusions:

• The engineered DelIscB system (enDelIscB) offers significantly enhanced genome editing activity.
• enDelIscB-based tools, including nucleases and base editors, are efficient and suitable for in vivo applications.
• These miniature genome editing tools hold great promise for diverse therapeutic and research applications.