Generation of Isogenic Human iPS Cell Line Precisely Corrected by Genome Editing Using the CRISPR/Cas9 System.

Authors:
Grobarczyk B, Franco B, Hanon K, Malgrange B.
In:
Source: Stem Cells
Publication Date: (2015)
Issue: 11(5): 774-87
Research Area:
Stem Cells
Gene Expression
Cells used in publication:
Induced Pluripotent Stem Cell (iPS), human
Species: human
Tissue Origin:
Platform:
4D-Nucleofector™ X-Unit
Experiment
After dissociation of feeder-free iPS colonies with Accutase 2x 10e5 iPS cells were resuspended in 20µl supplemented P3 solution; 3 plasmids for genomic engineering were used: pLCas9 (Addgene plasmid #44719), pLsgRNA (Addgene plasmid #41824; Church\\\'s lab) and HDR template; 2µg of each plasmid/20µl sample; 4D Nucleofector X-unit program CB-150; transferred in KO-SR medium with ROCK-inhibitor.
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.