To construct peptidyl-prolyl cis-trans isomerase never in mitosis A-interacting 1 (Pin1) knockout C17.2 neural stem cell (NSC) lines using CRISPR/Cas9 technology, elucidate the impact of Pin1 deficiency on the proliferation capacity of embryonic NSCs, and explore the underlying molecular mechanisms based on transcriptome sequencing.

Specific single guide RNAs (sgRNAs) targeting exon 2 of the mouse Pin1 gene were designed to construct the CRISPR/Cas9 targeting vectors. These vectors were transfected into C17.2 NSCs, followed by G418 selection to obtain Pin1 knockout single-cell colonies. The knockout efficiency and stemness maintenance (Nestin expression) were validated by immunofluorescence and Western blot analyses. Cell proliferation was evaluated using BrdU incorporation assays and Ki67 immunofluorescence staining. Differentially expressed genes (DEGs) were screened via RNA-Seq and subjected to pathway enrichment analysis. The expression of DEGs was further validated by quantitative real-time PCR (qRT-PCR) and Western blot. Finally, DAPT, a γ-secretase inhibitor, was employed to block the Notch signaling pathway to verify its role in Pin1-mediated cell proliferation.

Three C17.2 cell clones with biallelic Pin1 knockout were successfully generated. Compared to wild-type cells, Pin1 knockout cells maintained positive Nestin expression, but exhibited significantly increased BrdU incorporation and Ki67-positive rates (P < 0.05), indicating a state of hyperproliferation. RNA-Seq and subsequent validation experiments demonstrated that Pin1 deletion resulted in the significant activation of the Notch signaling pathway, characterized by the marked upregulation of the Notch1 protein and its downstream target gene Hes1 (P < 0.05). Treatment with DAPT not only effectively inhibited the hyperactivation of the Notch1 pathway in Pin1 knockout cells but also significantly reduced their proliferation rate.

Pin1 functions as a negative regulator in C17.2 NSCs, restricting excessive cellular proliferation by inhibiting the Notch1 signaling pathway. These findings reveal a novel mechanism by which Pin1 maintains proliferation homeostasis during the development of embryonic NSCs.

To knock out PIN1 gene of porcine fetal fibroblasts (PFFs) by clustered regular interval short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system in order to establish PIN1 knockout porcine fetal fibroblast cell lines.

Firstly, the homology of the PIN1 gene between humans and pigs was analyzed. Secondly, 2 sgRNAs targeting the second exon region of the porcine PIN1 gene were designed using online tools (http://crispor.tefor.net/) and then cloned into the pX330 (addgene #42230) skeleton plasmid. Finally, the PIN1 targeting plasmid and Neomycin resistant plasmid were co-transfected into PFFs. G418 screening was used to obtain resistant single-cell colonies. Sanger sequencing, qRT-PCR, and Western blotting were adopted to determine the PIN1 genotypes and expression levels, respectively.

The bioinformatics analysis showed that the amino acid sequence consistency and similarity of human and pig PIN1 proteins were 98%, and the root-mean-square deviation (RMSD) value of their 3-dimensional structures was 0.014. Fifteen homozygous monoclonal cell lines with PIN1 gene knockout were obtained, and the PIN1 protein was disrupted entirely in those PIN1 knockout colonies at translational level.

The PIN1 gene is efficiently knocked out in PFFs by a double sgRNAs-guided CRISPR/Cas9 system, and the porcine fetal fibroblast cell lines with PIN1 gene knocked out are successfully established.