Evaluation Of Different CRISPR/Cas9-based Gene Editing Systems To Study Specific Alleles Within LGALS3, A Gene Previously Associated With The Risk Of Various Types Of Cancer.

Galectin-3 is a glycoprotein of 31KDa with a chimeric structure encoded by LGALS3 gene located on chromosome 14. Galectin-3 is involved in several types of cancer including gastric, cervical, breast and colorectal carcinoma. Specifically, the single nucleotide polymorphism (SNP) rs4644 (c.191C>A, p.Pro64His) has been associated with the risk of developing these cancers. Proline 64 is broadly conserved across phylogenetically distant organisms. Histidine 64 is predicted to be damaging according to the analysis performed with Polyphen and SIFT. The aim of this study was to create isogenic cell lines that differ only for a single nucleotide, permitting further studies about the function of the polymorphism in different cancers. In this work, TPC-1 (thyroid cancer) cell lines, HCT-116 +/+ (colorectal cancer) cell lines and Nthy-Ori (non tumoral thyroid) cell lines were employed. The generation of the isogenic cell lines was performed with a Clustered Regularly Interspaced Short Palindromic Repeats-associated Endonuclease 9 (CRISPR/Cas9) system. Different vectors and approaches for DNA strand breaks induction (classical and double nickase strategies), as well as alternative strategies of delivery (chemical agent and lentivirus system) as well as different methods to perform the repair (single-stranded DNA oligonucleotides and donor vector), were employed and compared to each other in order to identify the most effective in terms of specificity, time, and costs. Isogenic cell lines were obtained following gene editing, with variable results depending on the strategy employed. Further steps will include the identification of possible off targets and the evaluation of how global gene expression is affected by proline to histidine variation. In the future these cells will be helpful to understand the biological role of this aminoacid change within Galectin-3.