An efficient and adaptable workflow for editing disease-relevant single nucleotide variants using CRISPR/Cas9 [article]

Inga Usher, Lorena Ligammari, Sara Ahrabi, Emily Hepburn, Calum Connolly, Gareth L Bond, Adrienne M Flanagan, Lucia Cottone
2021 bioRxiv   pre-print
Single nucleotide variants are the commonest genetic alterations in the human genome. At least 60,000 have been reported to be associated with disease. The CRISPR/Cas9 system has transformed genetic research, making it possible to edit single nucleotides and study the function of genetic variants in vitro. While significant advances have improved the efficiency of CRISPR/Cas9, the editing of single nucleotides remains challenging. There are two major obstacles: low efficiency of accurate
more » ... and the isolation of these cells from a pool of cells with other editing outcomes. We present data from 85 transfections of induced pluripotent stem cells and an immortalised cell line, comparing the effects of altering CRISPR/Cas9 design and experimental conditions on rates of single nucleotide substitution. We targeted variants in TP53, which predispose to several cancers, and in TBXT which is implicated in the pathogenesis of the bone cancer, chordoma. We describe a scalable and adaptable workflow for single nucleotide editing that incorporates contemporary techniques including Illumina MiSeq sequencing, TaqMan qPCR and digital droplet PCR for screening transfected cells as well as quality control steps to mitigate against common pitfalls. This workflow can be applied to CRISPR/Cas9 and other genome editing systems to maximise experimental efficiency.
doi:10.1101/2021.11.12.467071 fatcat:ki5zdfpe3vchfpz7ykec3f5pdm