Derivation of a CRISPR genome edited stem cell line containing a risk variant in TTC3.

Abstract

We identified a rare, nonsynonymous variant in the tetratricopeptide repeat domain 3 (TTC3) gene that segregated in all 11 Alzheimer disease (AD) individuals in a non-Hispanic white late onset Alzheimer disease (LOAD) family (mean AAO=75.7 years, Kohli, et al, 2016). This missense alteration, rs377155188 (p.S1038C), is predicted to be deleterious by five in silico algorithms and extremely rare in the gnomAD database (allele frequency=3.231x10-5 ). Studies have reported that cortical TTC3 expression is reduced in LOAD patients and negatively correlated with the AD neuropathology. To understand the mechanism by which the TTC3 alteration may contribute to LOAD risk, CRISPR/Cas9 genome edited induced pluripotent stem cells (iPSCs) were developed to examine cellular consequences in neuronal cells, which can otherwise only be collected postmortem. Starting with an iPSC line derived from a healthy 45 year old male donor of European ancestry, ASE-9203, genome editing was performed by introducing an sgRNA cloned into the Cas9 containing TLCV2 plasmid (Addgene) simultaneously with a ssODN template. An independent homozygous edited iPSC clone was isolated and verified by Sanger sequencing to carry the TTC3 variant, thus creating an isogenic pair of iPSCs. The stem cells were differentiated into forebrain neurons for 70 days and evaluated. Quantitative PCR analysis demonstrated that TTC3 levels were decreased in edited compared to unedited iPSCs, as well as day 30 and day 70 differentiated neurons. Since there is evidence that modulation of TTC3 affects neurite growth (Berto, et al, 2007), morphological measures of axon formation were assessed in differentiating neuronal cultures using the Incucyte Zoom. Preliminary studies demonstrate an increase in neurite outgrowth, which phenotypically corresponds with previous studies of a decrease in TTC3 function. Levels of amyloid beta 40 and 42 released from day 65 edited and unedited neurons were not significantly different. Results suggest that the TTC3 p.S1038C variant causes a loss of function. Utilizing a CRISPR genome edited iPSC carrying a homozygous alteration in TTC3 will enable us to determine how this genetic alteration which may contribute to specific cellular phenotypes and, on a broader scale, LOAD risk.