Simultaneous reprogramming and gene editing of human fibroblasts (Version 1.0)

Version

1.0

Notice

This page is the corresponding protocol tomestone page generated as part of the ATLAS-D2K shutdown in July 2025. Many links on this page may be broken.

Authors

Melissa H. Little; Sara Howden; James A. Thomson

Keywords

[‘CRISPR-Cas9 genome editing’, ‘induced pluripotent stem cells (iPSC)’, ‘Mutagenesis’, ‘Reprogramming’]

Subjects

[‘Genetic engineering’, ‘Genomics’]

Release Date

2019-02-15

Abstract

NOTE: This protocol is published online (Nat Protoc. 2018 May;13(5):875-898). Now available at PubMedCentral: PMCID: PMC5997775

The utility of human induced pluripotent stem cells (iPSCs) is enhanced by an ability to precisely modify a chosen locus with minimal impact on the remaining genome. However, the derivation of gene-edited iPSCs typically involves multiple steps requiring lengthy culture periods and several clonal events. Here, we describe a one-step protocol for reliable generation of clonally derived gene-edited iPSC lines from human fibroblasts in the absence of drug selection or FACS enrichment. Using enhanced episomal-based reprogramming and CRISPR/Cas9 systems, gene-edited and passage-matched unmodified iPSC lines are obtained following a single electroporation of human fibroblasts. To minimize unwanted mutations within the target locus, we use a Cas9 variant that is associated with decreased nonhomologous end-joining (NHEJ) activity. This protocol outlines in detail how this streamlined approach can be used for both monoallelic and biallelic introduction of specific base changes or transgene cassettes in a manner that is efficient, rapid (∼6-8 weeks), and cost-effective.

Procedure

Please see published protocol: PMID: 29622803 - PMC5997775

Associated_Publications

Howden SE, Thomson JA, Little MH. Simultaneous reprogramming and gene editing of human fibroblasts. Nat Protoc. 2018 May;13(5):875-898. PMCID: PMC5997775

Consortium

(Re)Building a Kidney (RBK) Consortium