Creating isogenic iPSC-based in vitro models for ASH1L-linked intellectual disability

• Main Author: Forsén, Robin
• Other Authors: Helsingin yliopisto, Bio- ja ympäristötieteellinen tiedekunta, University of Helsinki, Faculty of Biological and Environmental Sciences, Helsingfors universitet, Bio- och miljövetenskapliga fakulteten
• Format: Master Thesis
• Language: English
• Published: Helsingin yliopisto 2024
• Subjects: iPSC; neurodevelopmental disorders; ASH1L; CRISPR-based genome editing; in-vitro disease modelling; Genetiikka ja genomiikka; Genetics and genomics; Genetik och genomik; Genetiikan ja molekulaaristen biotieteiden maisteriohjelma; Master's Programme in Genetics and Molecular Biosciences; Magisterprogrammet i genetik och molekylära biovetenskaper; Northern Finland
• Online Access: http://hdl.handle.net/10138/577736

ASH1L is a Histone lysine methyltransferase belonging to the KMT family, which plays an important role in epigenetic gene regulation during development, and has been linked to neurodevelopmental disorders (NDDs). Mutations in ASH1L have been linked to NDDs including intellectual disability, autism spectrum disorder and Tourette’s syndrome. Induced pluripotent stem cell (iPSC) based models in combination with CRISPR/Cas9 gene editing provide powerful tools for studying the genetic causes of NDDs. The broad aim of this thesis was the creation of genetically modified iPSC lines for modelling NDDs linked to ASH1L. Patient and healthy cell lines were obtained from the Northern Finland Intellectual Disability cohort. With the long-term goal of generating a model by which to understand the impact of genetic background on reported causative mutations, CRISPR/Cas9-based genetic engineering was employed to correct the mutation in a patient cell line, and conversely, to generate a patient mutation in a healthy line. iPSC lines are known to be intrinsically variable and require thorough characterization of their genetic stability and pluripotency before use. Therefore, the secondary aim of this thesis was to subject newly reprogrammed iPSC lines to a battery of assays to first determine their suitability for downstream applications. Single-guide RNAs (sgRNAs) were designed to target a site ≤16 bp from the edit site. Single-stranded oligodeoxynucleotides (ssODNs) were used as HDR templates, incorporating the mutation of interest and 3-4 silent mutations to prevent binding by sgRNA after successful HDR. The Cas9-sgRNA complex and HDR template were introduced into the cell by nucleofection. Both mutations are frameshift mutations and are predicted to cause loss of function. Editing efficiency was evaluated with a T7E1 assay after nucleofection. Individual clones were isolated and MiSeq was used to sequence the region to a read depth of >1000reads per clone around the edit site to identify successful edits in these clones ...