Abstract: The mouse gene,S100A9 was used as an example to illustrate the expected sgRNA design. After in vitro transcription and Cas9 mRNA were injected into the fertilized egg by micro-injection,the embryos in the blastocyst stage were selected,and then performed with genotype identification. The results showed that a total of 113 fertilized eggs were microinjected,of which 28 successfully developed into the blastocyst stage,and five blastocysts produced effective genetic mutations at specific sites. The editing efficiency was 18%. This method is simple to operate,can rapidly verify the sgRNA activity in vivo,and provides a basis for the preparation of subsequent genetically edited mice.

Key words: CRISPR/Cas9, mouse, sgRNA validity, blastosphere