Optimization of a CRISPR/Cas9-mediated Knock-in Strategy at the Porcine Rosa26 Locus in Porcine Foetal Fibroblasts

Abstract

Genetically modified pigs have important roles in agriculture and biomedicine. However, genome-specific knock-in techniques in pigs are still in their infancy and optimal strategies have not been extensively investigated. In this study, we performed electroporation to introduce a targeting donor vector (a non-linearized vector that did not contain a promoter or selectable marker) into Porcine Foetal Fibroblasts (PFFs) along with a CRISPR/Cas9 vector. After optimization, the efficiency of the EGFP site-specific knock-in could reach up to 29.6% at the pRosa26 locus in PFFs. Next, we used the EGFP reporter PFFs to address two key conditions in the process of achieving transgenic pigs, the limiting dilution method and the strategy to evaluate the safety and feasibility of the knock-in locus. This study demonstrates that we establish an efficient procedures for the exogenous gene knock-in technique and creates a platform to efficiently generate promoter-less and selectable marker-free transgenic PFFs through the CRISPR/Cas9 system. This study should contribute to the generation of promoter-less and selectable marker-free transgenic pigs and it may provide insights into sophisticated site-specific genome engineering techniques for additional species.

Generation of Transgenic Cloned Buffalo Embryos Harboring the EGFP Gene in the Y Chromosome Using CRISPR/Cas9-Mediated Targeted Integration

“…The selection of positive cells using drugs is a very crucial method for obtaining transgenic cells to produce transgenic animals (68,(86)(87)(88). Although the efficiency at which insertion/deletions are introduced by the CRISPR/Cas9 system is very high (54,89), the knock-in efficiency still cannot satisfy the demands of agricultural production, which significantly limits the application of this technique (55). As has been indicated in many reports, drug selection can irreversibly harm the cells, the cells surviving drug screening had a decreased survival rate after multiple cell passages (55,88).…”

“…Although the efficiency at which insertion/deletions are introduced by the CRISPR/Cas9 system is very high (54,89), the knock-in efficiency still cannot satisfy the demands of agricultural production, which significantly limits the application of this technique (55). As has been indicated in many reports, drug selection can irreversibly harm the cells, the cells surviving drug screening had a decreased survival rate after multiple cell passages (55,88). DNA methylation and histone acetylation are important epigenetic events, the variations in the expression profile of epigenetic related genes in embryos and cloned animals are suggested to be linked with the reprogramming process (90)(91)(92).…”

“…At present, CRISPR/Cas9 mediated HDR is the commonly used approach to achieve precise and targeted integration of transgenes because of its high efficiency. There are some reports that the length of the homology arm (HA) can dramatically influence the efficiency of targeted integration of exogenous genes (55,56). Therefore, it may be possible to further improve the efficiency of CRISPR/Cas9-mediated HDR in the cells by optimizing HA length.…”

Development of whole-porcine monoclonal antibodies with potent neutralization activity against classical swine fever virus (CSFV) from single B cells

“…Next, 200 μl of fresh medium was added, and the cells were incubated at 37°C for 72 h. Subsequently, the cells were fixed with 80% cold acetone at -20°C overnight. The next day, the cells were washed and incubated with porcine CSFV-positive serum (1:100 dilution in PBS containing 10% FBS) at 37°C for 2 h. After being washed three times with PBS, FITC-labelled goat anti-pig IgG (Sigma-Aldrich, America) was added into each well, and the plates were incubated at 37°C for 0.5 h (55). Images were obtained using fluorescence microscopy (Olympus BX51).…”

CRISPR/Cas9-mediated integration of large transgene into pig potential safe harbor

“…The pig lines in our study can efficiently express the foreign gene that can be stably inherited to the offspring, and showed normal and healthy performance regardless of heterozygote or homozygote, which indicates CEP112 has the potential as a safe site for the expression of foreign genes like the ROSA26 locus that is frequently used for creating modified animals to achieve ubiquitous or conditional gene expression in vivo (Li et al . 2014; Xie et al . 2017).…”