Abstract:
: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. Of particular interest are the spectrum of autism disorders, some of which are monogenic such as Timothy syndrome (TS); others are multigenic such as the microdeletion and microduplication syndromes of the 16p11.2 chromosomal locus. Here, we report engineered human embryonic stem cell (hESC) lines for modeling these two disorders using locus-specific endonucleases to increase the efficiency of homology-directed repair (HDR). 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.
journal_name
Nucleic Acids Resjournal_title
Nucleic acids researchauthors
Martinez RA,Stein JL,Krostag AR,Nelson AM,Marken JS,Menon V,May RC,Yao Z,Kaykas A,Geschwind DH,Grimley JSdoi
10.1093/nar/gkv164subject
Has Abstractpub_date
2015-05-26 00:00:00pages
e65issue
10eissn
0305-1048issn
1362-4962pii
gkv164journal_volume
43pub_type
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