Study of the CRISPR/Cas3 System for Xenotransplantation.

ABSTRACT

The CRISPR/Cas3 system, classified in class I system, was recently focused as a new technology. For application of this system to porcine cells, the plasmids of bpNLS-Cascade, BPNLS-hCas3, and pBS-U6icrRNA were prepared. Initially, 2 crRNAs were established in the exon 9 of pig Gal-T (GGTA1) as #45 and #86. Next, hCas3 + #45 + #86 (group 1, control), Cascade + hCas3 + #45 (group 2), Cascade + hCas3 + #86 (group 3), and Cascade + hCas3 + #45 + #86 (group 4) were set and transfected into pig fibroblasts. Transfected cells were analyzed for bulk expression of α1,3Gal epitope by fluorescence-activated cell sorting (FACS), using a GSI-B4 lectin 2 days after the transfection. As the results, changes of expression are observed in order of G4>G2>G3, indicating the effect of the Cas3 system. Therefore, the nested polymerase chain reaction (PCR) for target region of GGTA1 was performed. Next, the PCR products from each group were checked in blotting, and the products were placed into the cloning sit of TOPO vector and transformed into Escherichia coli. Sixteen colonies of each group were checked by PCR, and clones containing PCR product with slightly varying length were evaluated. The direct sequence of these PCR changes were demonstrated as 294 to 754 bp deletions. In conclusion, we confirmed the effect of the CRISPR/Cas3 system on pig cell, especially in xenotransplantation.