Cas12a mediates efficient and precise endogenous gene tagging via MITI: microhomology-dependent targeted integrations

Area of Science:

• Molecular Biology
• Genetics
• Biotechnology

Background:

• Cas9-mediated gene editing often results in imprecise DNA integration via non-homology end-joining.
• Existing methods struggle with accuracy for site-specific gene insertions and tagging of endogenous genes.

Purpose of the Study:

• To develop a precise site-specific gene insertion method using microhomology-dependent targeted integration (MITI).
• To enhance the accuracy of exogenous DNA integration and endogenous gene tagging compared to Cas9 HITI.

Main Methods:

• Developed the MITI strategy utilizing Cas12a for microhomology-dependent targeted integration.
• Employed negative selection and a four-CrRNA array targeting donor vectors and genomic sites.
• Validated MITI for precise insertion of external DNA and tagging of endogenous genes.

Main Results:

• The MITI method demonstrated higher knock-in accuracy than Cas9 HITI.
• Precise ligation at all junctions was achieved using the MITI strategy with optimized conditions.
• MITI facilitates accurate gene insertions and endogenous gene tagging.

Conclusions:

• The Cas12a-based MITI method offers a significant advancement in precision genome engineering.
• MITI expands the toolkit for precise gene editing applications.
• This method provides a reliable tool for various gene editing applications requiring high accuracy.