The method using humanized yeast assumes a substitution of a gene in yeast with a homologous human gene under study. The inserted human gene is supposed to complement the function of the yeast gene. This can be detected as a change in yeast strain's phenotype. Different variants of human gene can then be used for the substitution. Based on their ability to complement the yeast counterpart, we can asses whether these variants have a negative effect on the normal function of a gene. We wanted to test the capability of human gene ATG16L1 to complement the homologous yeast gene ATG16. Both genes are involved in the proces of autophagy, and mutations in gene ATG16L1 have been associated with an increased risk of Chron's disease. In case of successful complementation, our next goal was to asses whether mutations 220G>A (Asp74Asn) and 898A>G (Thr300Ala) in gene ATG16L1 are pathogenic. With the use of genome editing technique CRISPR/Cas9, we performed a gene substitution in the yeast deletion strain atg16Δ. We then performed a series of phenotypic tests on the humanized strain, which showed that ATG16L1 is not capable of complementing the ATG16 gene. Further identification of pathogenicity for different variants of gene ATG16L1 was therefor impossible.
|
