A simple and highly efficient method for multi-allelic CRISPR-Cas9 editing in primary cell cultures.

ABSTRACT:

Background

CRISPR-Cas9-based technologies have revolutionized experimental manipulation of mammalian genomes. None-the-less, limitations of the delivery and efficacy of these technologies restrict their application in primary cells.

Aims

To create an optimized protocol for penetrant, reproducible, and fast targeted genome editing in cell cultures derived from primary cells, using patient-derived glioblastoma stem-like cells (GSCs) and human neural stem/progenitor cells (NSCs) for proof-of-concept experiments.

Methods and results

We employed transient nucleofection of Cas9:sgRNA ribonucleoprotein complexes composed of chemically synthesized 2'-O-methyl 3'phosphorothioate-modified sgRNAs and purified Cas9 protein. Insertion-deletion mutation (indel) frequency and size d

SUBMITTER: Hoellerbauer P 

PROVIDER: S-EPMC7685144 | biostudies-literature | 2020 Oct

REPOSITORIES: biostudies-literature