title: Generation of Isogenic Human iPS Cell Line Precisely Corrected by Genome Editing Using the CRISPR/Cas9 System.
abstract: Genome engineering and human iPS cells are two powerful technologies, which can be combined to highlight phenotypic differences and identify pathological mechanisms of complex diseases by providing isogenic cellular material. However, very few data are available regarding precise gene correction in human iPS cells. Here, we describe an optimized stepwise protocol to deliver CRISPR/Cas9 plasmids in human iPS cells. We highlight technical issues especially those associated to human stem cell culture and to the correction of a point mutation to obtain isogenic iPS cell line, without inserting any resistance cassette. Based on a two-steps clonal isolation protocol (mechanical picking followed by enzymatic dissociation), we succeed to select and expand corrected human iPS cell line with a great efficiency (more than 2% of the sequenced colonies). This protocol can also be used to obtain knock-out cell line from healthy iPS cell line by the NHEJ pathway (with about 15% efficiency) and reproduce disease phenotype. In addition, we also provide protocols for functional validation tests after every critical step.
authors.affiliation: GIGA-Neurosciences, Developmental Neurobiology Unit, University of Liège, Liège, Belgium.
authors.forename: Benjamin
authors.initials: B
authors.lastname: Grobarczyk
authors.forename: Bénédicte
authors.initials: B
authors.lastname: Franco
authors.forename: Kevin
authors.initials: K
authors.lastname: Hanon
authors.forename: Brigitte
authors.initials: B
authors.lastname: Malgrange
mesh_terms: D018929:Cell Culture Techniques; D064112:Clustered Regularly Interspaced Short Palindromic Repeats / Q000235:genetics*; D059766:DNA End-Joining Repair / Q000235:genetics; D005818:Genetic Engineering / Q000379:methods; D016678:Genome / Q000235:genetics*; D057809:HEK293 Cells; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000502:physiology*; D017354:Point Mutation / Q000235:genetics