Efficient Production of Gene-Modified Mice using Staphylococcus aureus Cas9

Area of Science:

• Molecular Biology
• Genetics
• Gene Editing Technologies

Background:

• CRISPR/Cas systems are powerful tools for modifying genes in mouse zygotes.
• Streptococcus pyogenes Cas9 (SpCas9) has been widely used, but its PAM (5'-NGG-3') requirement limits targetable sites.
• Staphylococcus aureus Cas9 (SaCas9) offers an alternative with a different PAM (5'-NNGRRT-3') and smaller size.

Purpose of the Study:

• To evaluate the efficiency and specificity of SaCas9 for genome editing in mouse zygotes.
• To demonstrate SaCas9's capability for gene knockout and knock-in applications.
• To explore SaCas9's potential for multiplex gene editing in preimplantation embryos.

Main Methods:

• Co-injection of SaCas9 mRNA and guide RNAs (gRNAs) targeting specific genes (Slx2, Zp1, Tyr) into mouse zygotes.
• Assessment of gene disruption via SaCas9-mediated cleavage.
• Demonstration of precise knock-in by co-introducing a DNA oligo encoding a Flag tag.

Main Results:

• SaCas9 efficiently and specifically edited the X-linked gene Slx2 and the autosomal gene Zp1 in mouse zygotes.
• SaCas9-mediated disruption of the tyrosinase (Tyr) gene resulted in mosaic coat color in C57BL/6J mice.
• Multiplex targeting enabled simultaneous disruption of multiple genes (Slx2, Zp1, Tyr).
• Precise knock-in of a Flag tag into the histone H1c gene was achieved.

Conclusions:

• SaCas9 is an effective tool for gene knockout and precise knock-in in mouse zygotes.
• SaCas9 expands the range of targetable sites compared to SpCas9 due to its distinct PAM preference.
• SaCas9 holds significant potential for genome editing in preimplantation embryos and generating gene-modified animal models.