CRISPR/Cas9-Mediated Highly Efficient Gene Targeting in Embryonic Stem Cells for Developing Gene-Manipulated Mouse...

Area of Science:

• Genetics and Genomics
• Molecular Biology
• Developmental Biology

Background:

• CRISPR/Cas9 facilitates direct genome editing in mouse zygotes.
• Direct embryo editing for large DNA knock-in (KI) shows low efficiency.
• Gene targeting in embryonic stem cells (ESCs) offers advantages for generating genetically modified mice.

Purpose of the Study:

• To describe an optimized protocol for large DNA KI in ESCs using CRISPR/Cas9.
• To enable efficient generation of gene-manipulated mouse models.
• To overcome challenges in direct embryo editing for complex genetic modifications.

Main Methods:

• CRISPR/Cas9-mediated genome editing was applied to ESCs for large DNA knock-in.
• Gene-targeted ESCs were used for chimera mouse production.
• The protocol is optimized for knock-in of large DNA fragments (several kb).

Main Results:

• The optimized method achieves efficient large-size DNA knock-in in ESCs.
• This approach facilitates the generation of gene-manipulated mouse models.
• It provides an alternative to less efficient direct embryo editing methods.

Conclusions:

• CRISPR/Cas9-mediated gene targeting in ESCs is a robust method for large DNA knock-in.
• This strategy is advantageous for creating complex genetically modified mouse models, including those from strains with challenging embryos.
• The protocol enhances the efficiency and throughput of generating gene-manipulated mouse models.