Point mutation refers to a type of gene mutation where a single base pair is altered. Many human diseases are associated with point mutations. To construct models for such diseases, the corresponding gene sites in rats or mice can be mutated to provide animal models for studying the pathogenic mechanisms of these mutations.

1. Technical Process:

gRNA design → Donor DNA synthesis → Microinjection (EPS targeting) → Mouse identification

2. Technical Advantages:

(1) Models for diseases caused by amino acid mutations.

(2) Relatively high efficiency of CRISPR point mutations.

3. Successful Cases:

Case One: Inserting several bases into the rat genome to prematurely terminate the expression of the Rag2 gene, resulting in the absence of protein expression and the lack of T cells and B cells in the rat. 

Figure 1. Genotype analysis of Rag2 knockout rats. 

Figure 2. Analysis of antibody expression in F344RG rats, with virtually undetectable levels of immunoglobulins.

Case Two: Replacement of small fragment gene sequences in the mouse genome. Using the CRISPR/Cas9 system, a 10 bp sequence in the mouse genome was replaced with the human sequence, making the gene function closer to that of humans. 

Figure 3. Schematic diagram of multi-base mutations.

Case Three: Single base mutation in the mouse genome. Using CRISPR/Cas9 technology to prepare point mutation mice, by mutating the G base to an A base in the c-kit gene of severely immunodeficient mice, changing valine at the 831st position to methionine, the obtained NPG-RF mice can avoid radiation and directly accept transplants of human hematopoietic stem cells, etc. 

Figure 4. Schematic diagram of base mutation, first line: sequencing results of homozygous mutant mice, second line: sequencing results of heterozygous mutant mice.