Thursday September 23, 2021 - 17:05 to 17:40
CPISPR / Cas3 system for pig Gal-T (GGTA1)
Shuji Miyagawa1, Masahito Watanabe2, Kazuki Sato1, Shuhei Kogata1, Chiyoshi Toyama1, Kazunori Masahata1, Masahumi Kamiyama1, Hiroshi Eguchi1, Akira Maeda1, Kazuto Yoshimi3, Tomoji Mashimo3, Hiroomi Okuyama1, Hiroshi Nagashima2.
1Pediatric Surgery, Osaka University Graduate School of Medicine, Suita/Osaka, Japan; 2International Institute for Bio-Resource Research, Meiji University, Kawasaki, Japan; 3Institute of Medical Science, The University of Tokyo, Tokyo, Japan
Background: CPISPR/Cas3 system which is classified in Class I system, has recently focused as a new technology in addition to Class II systems, such as Cas9, Cas 12a and Cas14a. This class I system represents most of CRISPR system and is more widely spread than class II group in bacteria & archaea.
Methods: First, the plasmids bpNLS-Cascade, BPNLS-hCas3, pBS-U6-crRNA were prepared. Two crRNAs were established in the exon 9 of pig Gal-T (GGTA1) as #45 & #86.
Next, hCas3+#45+#86 (Group 1, control), Cascade+hCas3+#45 (Group 2), Cascade+hCas3+#86 (Group 3) & Cascade+hCas3+#45+#86(Group 4) were set and transfected into pig fetal fibroblasts (D3), using a Neon transfection system.
Transfected cells were analyzed for bulk expression of a1,3Gal epitope by FACS, using a GSI-B4 lectin two days after the transfection.
The nested PCR products from each group were checked by blotting, and the products were insurted into the cloning sit of TOPO vector and then transformed. The E. coli colonies in each group were checked by PCR, and colonies containing the PCR product slightly varying length of PCR products were assessed by the Sanger sequence using the second F-R primers.
Results: As the results, changes of expression are observed in the order of G4> G2> G3, indicating the effect of the Cas3 system.
Sixteen colonies of E. coli in each group were checked by PCR, and colonies containing PCR product with slightly varing length were evaluated by the Sanger sequence. The changes in length were then turned out for two clones of G2 & G3, and four clones in G4.
Direct sequence of these PCR changes were demonstrated as 754 bp deletion on G2, 370 bp & 294 bp deletions on G3, and 547bp deletion, 637bp deletion & 749bp deletion on G4.
Conclusion: We confirmed the effect of the CRISPR/Cas3 system using pig fibroblast cell, especially in the field of xenotransplantation.