Evaluación funcional de un gen de predisposición al cáncer de colon en una línea celular editada genéticamente mediante CRISPR/Cas9


Maestro Moral, Claudia


Colorectal cancer (CRC) is the third most diagnosed type of cancer in men and the second one in women. An important factor in the predisposition to colorectal cancer is the genetic inheritance. Hereditary CRC includes various syndromes classified according to their phenotypic characteristics (polyposis and non-polyposis syndromes) and causal genes. Following the sequencing of the exomes of three members of a family with mixed polyposis, several variants were selected based on their presence in all polyposis-affected family members, their frequency in population controls, their function, and their enrichment in cases compared to controls. Based on these criteria, PARP1 c.412C>T; p.Arg138Cys was selected as the potential cause of the increased risk of developing polyposis in the studied family. Four additional predicted pathogenic PARP1 variants were identified in other cohorts of polyposis patients. The objective of this study is to generate cellular models to test the functional impact of the identified PARP1 variants. The genetic editing tool CRISPR-Cas9 was used to generate a heterozygous PARP1 knock-out (KO) cell line and, subsequently, a homozygous KO cell line. The success of these editions was verified by sequencing the clones of each cell line, followed by expression analysis by quantitative and semi-quantitative RT-PCR, and Western Blot. Knock-in cell lines that include the variants under study in the heterozygous KO cells are currently being generated. Selection of the edited clones will be performed with the KASP genotyping assays that we have designed.



Semino Margrett, Carlos
Valle, Laura


IQS SE -  Undergraduate Program in Biotechnology