Abstract: This work aimed to compare the effects of different target regions on the editing outcomes of CRISPR/Cas9 technology by using next generation targeted sequence, and provide a technical reference for target screening during application of this technology. This work was carried out as follows:1) Mouse Pyk2 gene was used in this study, and 3 target regions were designed in the first exon of this gene, through plasmid construction, cell transfection and next generation targeted sequencing of the cell DNA, we found that the total gene editing (Indels) efficiency of different target regions was different(site1:32.0%;site2:7.9%;site3:69.5%), and the preference for gene repair were also different among different groups. For the same target region, the editing efficiency was more stable in 2 experimental replicates(31.8% vs 32.3% for site1;7.4% vs 8.4% for site2;71.3% vs 67.8% for site3), and the preference of gene editing was relatively conservative; 2) Through Pyk2 site1 sgRNA and Cas9 in vitro transcription, mouse embryos microinjection, embryo transfer and genotyping, we found that the gene editing preference of the above target region was reproducible in gene editing animals production; 3) The mutant mouse with a single base inserted in site1 target region was used, sgRNA with a different base from site1 in orginal target was desigend. For the Pyk2 site1 target region, we found that single base inserts on Cas9 cleavage site had a significant impact on the efficiency of gene editing in the target region (49.2% vs 0%) by mutated mouse genomic PCR and Cas9 enzyme digestion in vitro cleavage test. In conclusion, target selection has a significant impact on the outcome of CRISPR/Cas9 gene editing. The next generation targeted sequence can effectively screen the CRISPR/Cas9 gene editing target region, and also obtain optimal results in the model animal production. In addition, inserting single base to the target region has a significant effect on gene editing efficiency.