A non-inheritable maternal Cas9-based multiple-gene editing system in mice.

Area of Science:

• Molecular Biology
• Genetics
• Animal Biotechnology

Background:

• CRISPR/Cas9 gene editing allows for multiple gene modifications via zygote injection.
• Current methods often involve co-injection of Cas9 DNA/mRNA and guide RNAs (gRNAs), with limitations in simultaneous gene editing efficiency and reliable transgenic animal generation.

Purpose of the Study:

• To investigate the efficacy of non-inheritable maternal Cas9 (maCas9) protein for multi-gene editing in mouse zygotes.
• To develop a method for generating "Cas9 transgene-free" gene-modified mice.

Main Methods:

• Utilized maCas9 protein derived from transgenic (Tg) mice with systemic Cas9 overexpression.
• Employed maCas9 protein in zygotes from Tg oocytes and wild-type sperm, and in non-Tg zygotes carrying maCas9.
• Injected nine different gRNAs into mouse zygotes carrying maCas9 to assess simultaneous editing of nine target loci.

Main Results:

• maCas9 protein demonstrated efficient genome editing comparable to conventional zygote microinjection.
• Successfully created "Cas9 transgene-free" gene-modified mice using maCas9.
• Achieved simultaneous editing of nine target loci in mouse zygotes by injecting nine distinct gRNAs with maCas9.

Conclusions:

• maCas9 protein offers a viable and efficient alternative for multi-gene editing in mouse zygotes.
• This approach facilitates the production of genetically modified animals, including Cas9 transgene-free mice, for advanced genome engineering.