1. Generation of Isogenic Human iPS Cell Line Precisely Corrected by Genome Editing Using the CRISPR/Cas9 System
- Author
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Benjamin Grobarczyk, Brigitte Malgrange, Bénédicte Franco, and Kevin Hanon
- Subjects
Genetics ,Cancer Research ,DNA End-Joining Repair ,Genome ,Cas9 ,Induced Pluripotent Stem Cells ,HEK 293 cells ,Cell Culture Techniques ,Cell Biology ,Biology ,Genome engineering ,HEK293 Cells ,Genome editing ,Humans ,Point Mutation ,CRISPR ,Clustered Regularly Interspaced Short Palindromic Repeats ,Genetic Engineering ,Induced pluripotent stem cell ,Gene - 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.
- Published
- 2015
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