Increasing Knockin Efficiency in Mouse Zygotes by Transient Hypothermia

Area of Science:

• Molecular Biology
• Developmental Biology
• Gene Editing

Background:

• CRISPR-Cas9 gene editing relies on DNA repair pathways: homology-directed repair (HDR) and nonhomologous end-joining (NHEJ).
• HDR is favored during the S/G2 phases of the cell cycle, while NHEJ is active throughout.
• Previous strategies aimed to increase HDR by manipulating cell cycle progression.

Purpose of the Study:

• To investigate if transient cold shock can enhance HDR-mediated gene editing.
• To assess the impact of cold shock on HDR and NHEJ rates in stem cells and mouse embryos.
• To evaluate the efficiency of CRISPR-Cas9 gene editing with cold shock in zygotes for improved knockin outcomes.

Main Methods:

• Utilized reporter systems in mouse embryonic stem cells and human-induced pluripotent stem cells to monitor HDR and NHEJ rates.
• Applied transient cold shock to cells and mouse embryos post-CRISPR-Cas9 ribonucleoprotein and single-stranded oligodeoxynucleotide template delivery.
• Analyzed gene editing efficiency via genotyping of blastocysts and confirmed live births by targeting the tyrosinase gene.

Main Results:

• Transient cold shock significantly increased HDR rates while decreasing NHEJ rates in reporter cell lines.
• Cold shock treatment of mouse zygotes post-electroporation with CRISPR-Cas9 and repair template enhanced knockin efficiency.
• Gene editing efficiency improved at multiple genetic loci without adverse effects on embryonic development, leading to live births of edited offspring.

Conclusions:

• Transient cold shock is a simple and effective method to enhance homology-directed repair-mediated gene editing.
• This approach improves knockin efficiency in both pluripotent stem cells and early-stage embryos.
• Cold shock offers a robust strategy for optimizing gene editing outcomes in various applications.