关键词: 慢病毒, CRISPR/Cas9, 猪胎儿成纤维细胞, BMPR-IB, 脱靶, 细胞增殖

Abstract: 【Objective】 The aims of this study were to edit the BMPR-IB gene in pig fetal fibroblasts (PFF) via a lentivirus-mediated CRISPR/Cas9 genome editing technology, and to investigate its effects on expression of relevant functional genes in the bone morphogenetic proteins (BMPs) signaling pathway. 【Method】 Twenty one single-guide RNAs (sgRNAs) targeting the eighth exon of porcine BMPR-IB gene were designed by the online software http://crispr.mit.edu. The sgRNA sequence with the highest score was selected for annealing with its complementary sequence (including adapters), and then the double-stranded DNA was ligated into the linearized lentiCRISPR v2, with the aim to obtain targeting plasmid. The targeting plasmid was mixed with packing vectors psPAX2 and pCMV-VSV-G at 5﹕4﹕1 molar ratio, then used to produce the recombinant lentivirus in 293T cells. To generate the induction mixture, the lentivirus supernatant was filtered through 0.45 μm, mixed with equal volume of fresh PFF growth medium, and finally polybrene was added to a final concentration of 6 μg·mL^-1. PFF cells were infected in induction mixture and centrifuged at 1 000 g for 1 h at 32℃, then cultured in a 37℃ incubator for 3 days. Three days post-transfection cells were selected with 3.5 μg·mL^-1 puromycin for 6-7 days, and resistant clones targeting BMPR-IB were expanded. Targeting cells were screened first by T7E1 digestion, and then the PCR and PCR-TA cloning were performed to confirm correct targeting. Quantitative real-time PCR was performed to detect the expression levels of relevant functional genes in BMPs signaling pathway. Protein expression of the BMPR-IB gene was detected by Western blotting. Cell Counting Kit-8 (CCK-8) kit was used to measure the proliferation capacity of targeted cells and control group cells. 【Result】 Both T7E1 assay and PCR sequencing showed that the targeted region was successfully edited in targeted cells. TA cloning and sequencing revealed the desired insertion and deletion mutations in the targeted region, and the indels mutation rate was 70%. Moreover, only one off-target site (OTS) was detected among 20 potential ones, and an off-target rate of 10% was observed at this site. Quantitative real-time PCR results demonstrated that the expression levels of BMPR-IB, CylinD2, Cdk2 and Bcl2 genes were significantly (P＜0.01) down-regulated in edited cells compared with wild-type cells. Western blotting results showed that the expression level of BMPR-IB in targeted cell was 38% of that in wild type cells. Cell proliferation assay revealed that the proliferation capacity of targeted cells was significantly lower than that of wild-type PFF cells of the same generation(P＜0.01). Significant losses of proliferation capacity in targeted PFF cells were found following the cell passages (P5, P7 and P9); while there was no significant difference between passage 5 and passage 7 or 9 in control cells. This loss of proliferation capacity in targeted cells does not seem to be caused by the puromycin selection process, as control cells did not show the same loss when underwent the same process. 【Conclusion】 The lentivirus-mediated CRISPR/Cas9 system is efficient for targeted gene editing in PFF. The expression levels of relevant genes in BMPs signaling pathway were significantly down-regulated in BMPR-IB edited cells, indicating that BMPR-IB plays an important role in regulating the proliferation of PFF cells.

Key words: Lentivirus, CRISPR/Cas9, porcine fetal fibroblasts, BMPR-IB, off-target, cell proliferation