Establishment and characterization of cytochrome P450 1A1 CRISPR/Cas9 Knockout Bovine Foetal Hepatocyte Cell Line (BFH12)

Abstract

The cytochrome P450 1A (CYP1A) subfamily of xenobiotic metabolizing enzymes (XMEs) consists of two different isoforms, namely CYP1A1 and CYP1A2, which are highly conserved among species. These two isoenzymes are involved in the biotransformation of many endogenous compounds as well as in the bioactivation of several xenobiotics into carcinogenic derivatives, thereby increasing the risk of tumour development. Cattle (Bos taurus) are one of the most important food-producing animal species, being a significant source of nutrition worldwide. Despite daily exposure to xenobiotics, data on the contribution of CYP1A to bovine hepatic metabolism are still scarce. The CRISPR/Cas9-mediated knockout (KO) is a useful method for generating in vivo and in vitro models for studying xenobiotic biotransformations. In this study, we applied the ribonucleoprotein (RNP)-complex approach to successfully obtain the KO of CYP1A1 in a bovine foetal hepatocyte cell line (BFH12). After clonal expansion and selection, CYP1A1 excision was confirmed at the DNA, mRNA and protein level. Therefore, RNA-seq analysis revealed significant transcriptomic changes associated with cell cycle regulation, proliferation, and detoxification processes as well as on iron, lipid and mitochondrial homeostasis. Altogether, this study successfully generates a new bovine CYP1A1 KO in vitro model, representing a valuable resource for xenobiotic metabolism studies in this important farm animal species.

Keywords: Bovine; CRISPR/Cas9; CYP1A1; Knockout; Liver cells; Transcriptome.

Fig. 1

Description of CRISPR/Cas9 mediated CYP1A1 KO and confirmatory assays. (a) The two gRNAs (red arrows) were designed to direct Cas9 machinery in CYP1A1 promoter region and 3’ UTR. The Cas9 activity at these sites leads to a double-stranded break and a consequent deletion of ~ 5,173 bp. (b) Gene expression data (-fold change, arbitrary units, AU) are reported as the mean ± SEM of three biological replicates, each performed in duplicate. (c1) Cytochrome P450 1A1 immunoblotting, using β-actin as loading control. (c2) Densitometric analysis data are expressed in AU as the mean ± SEM of three biological replicates. (d) Cytochrome P450 1A1-dependent catalytic activity. The ethoxyresorufin O-deethylation (EROD) activity is expressed in nmoles min⁻¹ mg⁻¹ protein and represents the mean ± SEM of three biological replicates, each performed in duplicate. Statistical analysis: unpaired t-test with Welch’s correction. *: p < 0.05 and ***: p < 0.001, CYP1A1^KO vs. CYP1A1^CTL cells

Fig. 2

(a) Volcano plots representing the differentially expressed genes (DEGs) identified by comparing CYP1A1^KO and CYP1A1^CTL cells, using a FDR threshold of 0.05. (b, c) Focus on the gene expression of glutathione transferases (GSTs), epoxide hydrolase 1 (EPHX1) and members of the solute carrier (SLC) family of drug transporters. Data are expressed as the mean LogCPM ± SEM of three biological replicates

Fig. 3

GO (a) and KEGG (b) enrichment analysis of DEGs in CYP1A1^KO vs CYP1A1 cells. Gene ratio is the percentage of DEGs over the total number of genes in each pathway. Count (dot size) represents the number of DEGs enriched in a certain pathway. The colour gradient represents the adjusted significance level (p. adjusts), according to the Benjamin-Hochberg method