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

ABSTRACT: 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 ?-synuclein and present their comparative phenotypes.

SUBMITTER: Arias-Fuenzalida J 

PROVIDER: S-EPMC5830965 | biostudies-literature | 2017 Nov

REPOSITORIES: biostudies-literature