Genome engineering of isogenic human ES cells to model autism disorders

Nucleic Acids Research
Refugio A MartinezJoshua S Grimley

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 disorde...Continue Reading

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Citations

Dec 27, 2015·Current Opinion in Neurobiology·Zhexing WenGuo-li Ming
Oct 2, 2015·Autism Research : Official Journal of the International Society for Autism Research·Michael W NestorGene J Blatt
May 31, 2016·Stem Cells International·Piya PrajumwongsParinya Noisa
Jun 28, 2016·International Journal of Molecular Sciences·Sylvia Merkert, Ulrich Martin
Oct 19, 2016·Nature Reviews. Neuroscience·Prashanth RajarajanSchahram Akbarian
Dec 3, 2016·Regenerative Medicine·Kuang-Yui Chen, Paul S Knoepfler
Apr 23, 2020·Current Psychiatry Reports·Debamitra DasDimitrios Avramopoulos
Jul 22, 2019·Experimental Biology and Medicine·Dan HanGuoliang Li
Apr 14, 2017·Cellular and Molecular Life Sciences : CMLS·Arun Pandian ChandrasekaranSuresh Ramakrishna
Feb 12, 2017·Journal of Neuroscience Research·Zhexing Wen

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Methods Mentioned

BETA
transfection
PCR
electrophoresis
transgenic

Software Mentioned

UCSC genome browser
ENSEMBL
biomaRt
Biostrings
TALEN
R
HUGO
ssODA
BSgenome
TALENSeek

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