Evaluation of gene editing strategies based on CRISPR/Cas9 to study specific alleles of LGALS3 associated with the risk of papillary thyroid carcinoma

Galectin-3 is involved in thyroid carcinogenesis. The single nucleotide polymorphism (SNP) rs4644 (c.191C>A, p.Pro64His) within its encoding gene, LGALS3, is completely conserved across phylogenetically distant organisms, and it was predicted to be damaging by analysis performed with Polyphen and SIFT. Thus, we hypothesized that this SNP could affect the risk to develop DTC. The hypothesis was corroborated by a case-control association study (1155 DTC cases and 1222 controls), showing a protective role for the carriers of the Histidine-64 (OR=0.66; IC 95% 0.46- 0.93) versus the Proline-64 allele. To better understand the role of this SNP, cell lines underwent gene editing for altering the specific LGALS3 genotype. The “generation” of the 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, as well as alternative strategies of delivery, were employed and compared to each others in order to identify the most effective in terms of specificity, time, and costs. Isogenic cell lines were obtained following gene editing although with variable results, depending from the strategy employed. Further steps will include the identification of possible off targets and the evaluation of how global gene expression is affected by Pro to His variation. In the future these cells will be helpful to understand the biologic role of this aminoacid change within galectin-3.