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.
Highlights
CRISPR/Cas[9] is a powerful method for editing and introducing specific changes to the genome[1,2,3]
The only difference between the two donor plasmids is the inserted gene used for antibiotic selection, one using puromycin and the other blasticidin
Improved efficiency of homology-directed repair (HDR) homozygous change is required both for generation of experimental models of diseases which are caused by a homozygous mutation and for enabling potential new treatment avenues
Summary
CRISPR/Cas[9] is a powerful method for editing and introducing specific changes to the genome[1,2,3]. One of the most challenging applications of the technique is the introduction of bi-allelic changes, which is often necessary for either obtaining experimental models or for correction of disease-related recessive variants. Such editing of both alleles is required in case of retinal degenerative diseases, sickle cell disease, β-thalassemia, cystic fibrosis[4] and various mutations related to human cancer[5,6]. The application of gene editing in relation to introducing a desired homozygous change in cells could greatly benefit from developing efficient experimental approaches that are less time consuming and more specific. Prove the edited allele with puromycin resistant gene at correct region Prove the edited allele with blasticidin resistant gene at correct region
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