Efficient Cas9-based Genome Editing Using CRISPR Analysis Webtools in Severe Early-onset-obesity Patient-derived iPSCs.

The CRISPR system is an adaptive defense mechanism used by bacteria and archaea against viruses and plasmids. The discovery of the CRISPR-associated protein Cas9 and its RNA-guided cleavage mechanism marked the beginning of a new era in genomic engineering by enabling the editing of a target region in the genome. Gene-edited cells or mice can be used as models for understanding human diseases. Given its high impact in functional genomic experiments on different model systems, several CRISPR/Cas9 protocols have been generated in the past years. The technique uses a straightforward "cut and stitch" mechanism, but requires an accurate step-by-step design. One of the key points is the use of an efficient programmable guide RNA to increase the rate of success in obtaining gene-specific edited clones. Here, we describe an efficient editing protocol using a ribonucleotide protein (RNP) complex for homology-directed repair (HDR)-based correction of a point mutation in an induced pluripotent stem cell (iPSC) line generated from a 14-year-old patient with severe early-onset obesity carrying a de novo variant of ARNT2. The resulting isogenic iPSC line, named CUIMCi003-A-1, has a normal karyotype, expresses stemness markers, and can be differentiated into progenies from all three germ layers. We provide a detailed workflow for designing a single guide RNA and donor DNA, and for isolating clonal human iPSCs edited with the desired modification. This article also focuses on parameters to consider when selecting reagents for CRISPR/Cas9 gene editing after testing their efficiency with in silico tools.