Simultaneous targeting of linked loci in mouse embryos using base editing

Area of Science:

• Genetics
• Molecular Biology
• Genome Engineering

Background:

• Simultaneously mutating linked genetic loci is challenging with CRISPR/Cas9 due to DNA breaks causing deletions.
• Base editing offers a novel approach by directly converting base pairs without double-strand breaks, enabling precise linked mutations.

Purpose of the Study:

• To develop and demonstrate a base editing strategy for simultaneous introduction of mutations into linked loci.
• To analyze the function of linked cis-regulatory elements in gene activation.

Main Methods:

• Co-injection of two base editors and two single-guide RNAs (sgRNAs) into mouse zygotes.
• Introduction of C-to-T transitions into two cytokine-sensing transcription factor binding sites separated by 9 kb.
• Functional analysis of gene activation in mammary tissue during pregnancy and lactation.

Main Results:

• Successfully introduced simultaneous C∙G-to-T∙A base edits into linked transcription factor binding sites.
• Identified an enhancer that activates two flanking genes in mammary tissue.
• Demonstrated the potential of base editing for precise germline genome engineering.

Conclusions:

• Base editing overcomes limitations of CRISPR/Cas9 for simultaneous linked locus mutation.
• This method facilitates functional analysis of linked cis-elements and creation of disease models.
• The technique has broad implications for genetic research, disease modeling, and therapeutic applications.