FACS-Assisted CRISPR-Cas9 Genome Editing Facilitates Parkinson’s Disease Modeling

Arias-Fuenzalida J, Jarazo J, Qing X, Walter J, Gomez-Giro G, Nickels SL, Zaehres H, Schöler HR, Schwamborn JC
Source: Stem Cell Reports
Publication Date: (2017)
Issue: 14;9(5): 1423-1431
Research Area:
Basic Research
Cells used in publication:
Induced Pluripotent Stem Cell (iPS), human
Species: human
Tissue Origin:
4D-Nucleofector® X-Unit

Stem cell culture and electroporation.

The following human induced pluripotent stem (iPS) cells reprogrammed with non-integrative episomal methods were used: A13777 from female cord blood-derived CD34pos cells. Cell lines were cultured in Essential 8 medium on Geltrex or matrigel. Cells were normally dissociated with accutase and plated in media containing ROCK inhibitor Y27632 at 10µM for 24h after dissociation. Cells were subjected to positive selection with puromycin at a concentration of 0.5µg/mL. Cells were electroporated using 4D-Nucleofector System and a 4D kit for human dermal fibroblast (Lonza cat no. V4XP). Parental pre-electroporation line presents micro-duplication 20q11.21.


Genome editing and human induced pluripotent stem cells hold great promise for the development of isogenic disease models and the correction of disease-associated mutations for isogenic tissue therapy. CRISPR-Cas9 has emerged as a versatile and simple tool for engineering human cells for such purposes. However, the current protocols to derive genome-edited lines require the screening of a great number of clones to obtain one free of random integration or on-locus non-homologous end joining (NHEJ)-containing alleles. Here, we describe an efficient method to derive biallelic genome-edited populations by the use of fluorescent markers. We call this technique FACS-assisted CRISPR-Cas9 editing (FACE). FACE allows the derivation of correctly edited polyclones carrying a positive selection fluorescent module and the exclusion of non-edited, random integrations and on-target allele NHEJ-containing cells. We derived a set of isogenic lines containing Parkinson’s-disease-associated mutations in a-synuclein and present their comparative phenotypes