Prader-Willi syndrome (PWS) is a complex multisystemic genetic disorder with implications on the endocrine and neurologic systems, metabolism, and behaviour. PWS arises from the lack of expression of genes on the paternally derived chromosome 15q11.2-q13. Cases harbouring microdeletions narrowed the PWS critical region to the SNORD116 family, which is the primary genetic determinant of the PWS phenotype. SNORD116 gene cluster consists of 29 homologous genes that belong to the group of C/D-box small nucleolar RNAs. Since SNORD116 lacks obvious antisense elements against canonical RNA targets, its biological function remains largely unknown. For this reason, one of the most important tasks in this field of research is to identify the non-canonical RNA targets of SNORD116 that would significantly help determine the role of this gene cluster in the pathophysiology of the disease. Within this work, we wanted to create SNORD116 knock-out cell models using the scCRISPR method in NTERA2/D1 and SH-SY5Y cell lines. The cellular models would serve as a basis for further research and validation of the most probable potential targets of SNORD116. Firstly, we selected target sequences at the PWS locus and designed sgRNAs, which were then amplified and extended by PCR to obtain the appropriate components for the scCRISPR system. After we prepared and validated all components, we tried to optimize the transfection efficiency in both cell lines with various methods. Both plasmids and sgRNA constructs were then introduced into cells with the method that proved to be the most effective. To check if the scCRISPR system caused the desired deletion in target cells we analysed changes in genomic DNA at the PWS locus in individual isolated clones by PCR with specific primers. Unfortunately, the deletion of SNORD116 was unsuccessful in both of the selected cell lines, most likely due to low transfection efficiency. Despite the fact that we were not able to create the desired cell models, we successfully optimized the CRISPR/Cas9 system and methods needed for site-directed genome editing. In addition, we optimized methods to verify mutations and methods for the selection and generation of individual clones. We believe our work provides good basis for further knock-out experiments and thus generation of cell models that will help us discover the biological role of this enigmatic gene cluster.
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