Method for preparing CKO/KI animal model by using CAS9 technology

A method for preparing Nestin-Cre (Nes-Cre) model mice by using Cas9 technology, comprising the following steps:

• step 1:
• preparation of a Cas9 protein with nuclease activity for subsequent steps, wherein the Cas9 protein is prepared by expression and purification in vitro;
• step 2:
• screening of sgRNA, comprising:
• (1) designing a plurality of sgRNAs for targeting a Nes-Cre transgene to a knock-in locus and preparing transcription templates thereof, one of the plurality of sgRNAs being Nes-Cre-S2 consisting of a sequence as shown in SEQ ID NO:
• 4;
• (2) transcribing the sgRNA in vitro using a transcription kit, and the transcribed sgRNA being for later use;
• and
• (3) transferring the sgRNA from step 2(2) and the Cas9 protein from step 1 into mouse fertilized eggs by microinjection or electroporation, and testing the obtained embryos for sgRNA cleavage activity, thereby obtaining Nes-Cre-S2;
• step 3:
• construction of a single-stranded DNA targeting vector consisting of a sequence as shown in SEQ ID NO:
• 11, containing the Nes-Cre transgene;
• step 4:
• mouse embryo injection and transplantation, comprising:
• mixing the single-stranded DNA targeting vector constructed according to step 3, the Cas9 protein from step 1, and Nes-Cre-S2 from step 2, and carrying out mouse embryo injection and transplantation by using the mixed sample;
• and
• step 5:
• marking mice born after transplantation as F0 and carrying out the genotype identification of F0;
• breeding sexually mature F0 with the positive genotype identification, and marking the offspring mice thereof as F1;
• and analyzing and verifying the F1 mice, and the F1 mice with the positive genotype verification being the prepared Nes-Cre model mice.