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Stem Cell Reports. 2017 Sep 12;9(3):725-731. doi: 10.1016/j.stemcr.2017.07.003. Epub 2017 Aug 3.

Rapid Generation of Human Genetic Loss-of-Function iPSC Lines by Simultaneous Reprogramming and Gene Editing.

Author information

1
Department of Neurology, University of Michigan Medical School, 5021 BSRB, 109 Zina Pitcher Place, Ann Arbor, MI 48109-2200, USA.
2
Department of Pediatrics, University of Michigan Medical School, Ann Arbor, MI 48109, USA.
3
Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor, MI 48109, USA; Molecular and Behavioral Neuroscience Institute, University of Michigan Medical School, Ann Arbor, MI 48109, USA.
4
Department of Neurology, University of Michigan Medical School, 5021 BSRB, 109 Zina Pitcher Place, Ann Arbor, MI 48109-2200, USA; VA Ann Arbor HealthCare System, Ann Arbor, MI 48105, USA. Electronic address: parent@umich.edu.

Abstract

Specifically ablating genes in human induced pluripotent stem cells (iPSCs) allows for studies of gene function as well as disease mechanisms in disorders caused by loss-of-function (LOF) mutations. While techniques exist for engineering such lines, we have developed and rigorously validated a method of simultaneous iPSC reprogramming while generating CRISPR/Cas9-dependent insertions/deletions (indels). This approach allows for the efficient and rapid formation of genetic LOF human disease cell models with isogenic controls. The rate of mutagenized lines was strikingly consistent across experiments targeting four different human epileptic encephalopathy genes and a metabolic enzyme-encoding gene, and was more efficient and consistent than using CRISPR gene editing of established iPSC lines. The ability of our streamlined method to reproducibly generate heterozygous and homozygous LOF iPSC lines with passage-matched isogenic controls in a single step provides for the rapid development of LOF disease models with ideal control lines, even in the absence of patient tissue.

KEYWORDS:

CRISPR/Cas9; epileptic encephalopathy; gene editing; genetic epilepsy; induced pluripotent stem cells; reprogramming

PMID:
28781079
PMCID:
PMC5599229
DOI:
10.1016/j.stemcr.2017.07.003
[Indexed for MEDLINE]
Free PMC Article

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