CRISPR/Cas9-mediated one step bi-allelic change of genomic DNA in iPSCs and human RPE cells <i>in vitro</i> with dual antibiotic selection

Supharattanasitthi, Wasu, Carlsson, Emil, Sharif, Umar and Paraoan, Luminita ORCID: 0000-0001-7568-7116 (2019) CRISPR/Cas9-mediated one step bi-allelic change of genomic DNA in iPSCs and human RPE cells <i>in vitro</i> with dual antibiotic selection. SCIENTIFIC REPORTS, 9 (1). 174-.

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Official URL: http://dx.doi.org/10.1038/s41598-018-36740-2

Abstract

CRISPR/Cas9 causes double-stranded DNA breaks that can undergo DNA repair either via non-homologous end joining (NHEJ) or, in the presence of a template, homology-directed repair (HDR). HDR is typically used to insert a specific genetic modification into the genome but has low efficiency compared to NHEJ, which is lowered even further when trying to create a homozygous change. In this study we devised a novel approach for homozygous single base editing based on utilising simultaneously two donor DNA templates cloned in plasmids with different antibiotic resistant genes. The donor templates were co-transfected alongside the CRISPR/Cas9 machinery into cells and a double antibiotic selection was optimised and allowed the isolation of viable desired clones. We applied the method for obtaining isogenic cells homozygous for variant B cystatin C, a recessive risk factor for age-related macular degeneration and Alzheimer's disease, in both induced Pluripotent Stem Cells (iPSCs) and a human RPE cell line. Bi-allelic gene edited clones were validated by sequencing, demonstrating that the double antibiotic templates approach worked efficiently for both iPSCs and human differentiated cells. We propose that this one step gene editing approach can be used to improve the specificity and frequency of introducing homozygous modifications in mammalian cells.

Item Type:	Article
Uncontrolled Keywords:	Cell Line, Epithelial Cells, Humans, DNA, Cystatin C, Induced Pluripotent Stem Cells, CRISPR-Cas Systems, Gene Editing
Depositing User:	Symplectic Admin
Date Deposited:	27 Nov 2018 10:01
Last Modified:	19 Oct 2023 08:48
DOI:	10.1038/s41598-018-36740-2
Open Access URL:	https://www.nature.com/articles/s41598-018-36740-2
Related URLs:	Author Publisher
URI:	https://livrepository.liverpool.ac.uk/id/eprint/3029074