Genome engineering of isogenic human ES cells to model autism disorders.
Bottom Line: Isogenic pluripotent stem cells are critical tools for studying human neurological diseases by allowing one to study the effects of a mutation in a fixed genetic background.We developed a system to: (1) computationally identify unique transcription activator-like effector nuclease (TALEN) binding sites in the genome using a new software program, TALENSeek, (2) assemble the TALEN genes by combining golden gate cloning with modified constructs from the FLASH protocol, and (3) test the TALEN pairs in an amplification-based HDR assay that is more sensitive than the typical non-homologous end joining assay.We applied these methods to identify, construct, and test TALENs that were used with HDR donors in hESCs to generate an isogenic TS cell line in a scarless manner and to model the 16p11.2 copy number disorder without modifying genomic loci with high sequence similarity.
Affiliation: Allen Institute for Brain Science, Seattle, WA 98103, USA.Show MeSH
Related in: MedlinePlus
Mentions: TALENSeek first creates the super-isoform, the furthest 5′ and 3′ length across all isoforms defined in ENSEMBL, for the specified gene (Figure 1a). Near the transcriptional start and stop sites, an acceptable TALEN-binding site pair is sought where the pairs are separated by 15–30 bp and each 14–19 bp binding site begins with a T. The program searches iteratively into the gene until an acceptable site is found (Figure 1b and c). This site is then searched for uniqueness in the genome, allowing for a number of user-defined mismatches. If a homologous sequence in human and mouse is specified, the binding site identified in the human genome is tested to be within 250 bp of the start or end site of that same gene in the mouse genome (Figure 1d). The user has the option to test if the TALEN-binding site is unique in each genome. The output includes a .bed file for visualization in the UCSC genome browser (38) that identifies the individual TALEN-binding sites in the human and mouse genomes for each specified target (Figure 1e).
Affiliation: Allen Institute for Brain Science, Seattle, WA 98103, USA.