Gene targeting on CYP2C locus in rats using the CRISPR/CAS9 system

Cytochrome P450 (CYP) is a heme-containing enzyme superfamily, which plays a significant role in the metabolism of many endogenous and exogenous compounds, such as clinical drugs, pollutants in the environment, carcinogens and fatty acids. CYP2C cluster, in humans, is one of the most important CYP subfamilies, which accounts for about 25% of all CYP-involved hepatic drug transformation. CYP2C isoforms also metabolize endogenous substrates, such as arachidonic acid and some steroids. Moreover, CYP2C9 has been implicated to participate in the regulation of vascular tone. The expression of CYP2C8 and CYP2C9 is found in breast tumor tissues, and their polymorphisms may influence breast tumor characteristics and disease-free survival in tamoxifen treated patients. To understand more about roles of CYP2C clusters in drugs and human diseases, we tried to generate Cyp2c (Cyp2c6, Cyp2c11 and Cyp2c12) knockout rat model by using CRISPR/Cas9 system. We got two F0 founders, which contained frameshift mutations at both Cyp2c11 and Cyp2c12 loci. And the two F0 offsprings were crossed with wild type rats to generate heterozygotes of both Cyp2c11 and Cyp2c12 in one progeny. This chapter described the construction of Cyp2c6/11/12 knockout rat model by CRISPR/Cas9. Furthermore, we discussed the gains and lessons in generating this gene-targeting rat model and proposed our strategy to generate a rat model lacking all CYP2C isoforms.