Generating Loss-of-function iPSC Lines with Combined CRISPR Indel Formation and Reprogramming from Human Fibroblasts

Andrew M Tidball; Preethi Swaminathan; Louis T Dang; Jack M Parent

doi:10.21769/BioProtoc.2794

Generating Loss-of-function iPSC Lines with Combined CRISPR Indel Formation and Reprogramming from Human Fibroblasts

Bio Protoc. 2018 Apr 5;8(7):e2794. doi: 10.21769/BioProtoc.2794.

Authors

Andrew M Tidball¹, Preethi Swaminathan¹, Louis T Dang², Jack M Parent^{1

3}

Affiliations

¹ Department of Neurology, University of Michigan Medical School, Ann Arbor, MI, USA.
² Department of Pediatrics, University of Michigan Medical School, Ann Arbor, MI, USA.
³ VA Ann Arbor HealthCare System, Ann Arbor, MI, USA.

Abstract

For both disease and basic science research, loss-of-function (LOF) mutations are vitally important. Herein, we provide a simple stream-lined protocol for generating LOF iPSC lines that circumvents the technical challenges of traditional gene-editing and cloning of established iPSC lines by combining the introduction of the CRISPR vector concurrently with episomal reprogramming plasmids into fibroblasts. Our experiments have produced nearly even numbers of all 3 genotypes in autosomal genes. In addition, we provide a detailed approach for maintaining and genotyping 96-well plates of iPSC clones.

Keywords: CRISPR/Cas9; Cellular reprogramming; Disease modeling; Genome editing; Human fibroblasts; Induced pluripotent stem cells.

Abstract

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