Life Technologies

30 Life Technologies ™ | Parkinson's cell models Summary, conclusions, and next steps In the work shown here, we generated a panel of isogenic iPSC lines using GeneArt ® Precision TALs technology. From the parental MSA donor iPSC line, we created two derivative cell lines that exhibited either a reduced expression, or no expression, of α-synuclein. We expect that this set of cell lines will be useful not only in the establishment of disease models for MSA, but also to better understand the impact of α-synuclein levels on disease-relevant phenotypes. Starting from a second parental cell line that contained mutations in both the LRRK2 and GBA genes, we independently corrected each mutation back to wild type to better understand how synergies between these mutations may impact disease-relevant phenotypes. Future work could include the generation of a cell line in which both genes were corrected back to a wild type state. In addition to the TAL-based editing methods used here, other editing methods have recently garnered much attention based on their potential advantages in terms of cost and ease of use. The use of CRISPR-based technologies is of particular interest due to the simple design of the gene-targeting construct (the cleaving enzyme is guided directly to the site of interest by a relatively short strand of RNA that provides an exact complementary match to the target), which is substantially more economical to generate than a pair of large TAL constructs. Additionally, CRISPR-based editing has been reported to show greatly enhanced efficiency of cleavage of the target locus [7]. In fact, when we performed exploratory experiments using the CRISPR system to target the SNCA locus, over half of the colonies surveyed showed the presence of cleavage at that locus. Given that the extent of off-target effects for current CRISPR technology has yet to be defined and the importance of having true isogenic lines for our studies, we decided to focus on using the TAL-based system for the creation of the cell lines. To add confidence that the lines we generated are truly "isogenic", we performed full-exome sequencing on the parental MSA donor line and its two offspring lines, to screen for the presence of any indels caused by off-target cleavage. Using a modification of a bioinformatics workflow that was designed to compare tumor samples to normal samples, we saw no emergent indels across any of the exons in any of the lines, except for at specifically targeted sites within the SNCA gene. Because the homologous recombination used to introduce the LRRK2 and GBA edits can also be accompanied by errors, we are further investigating these lines for such effects. Our current efforts are focused on comparing phenotypic differences between the lines we created. While we previously described the development of assays that can be performed at the neural stem cell (NSC) stage, our ultimate goal is to differentiate these cells into midbrain dopaminergic neurons for phenotypic comparison. In the work described here, we developed an optimized TAL-based editing workflow for iPSCs that utilizes three genomic analysis methods for colony screening to efficiently identify edited clones. We also utilized exome sequencing to screen for and analyze off-target effects. We hope to share our protocols and insights to enable the broader research community to successfully perform TAL-based genome editing of iPSCs to develop relevant models with which to study—and perhaps one day cure— diseases such as Parkinson's disease.

• Cover