Efficient Multi-Allelic Genome Editing of Primary Cell Cultures via CRISPR-Cas9 Ribonucleoprotein Nucleofection.

Abstract

CRISPR-Cas9-based technologies have revolutionized experimental manipulation of mammalian genomes. However, limitations regarding the delivery and efficacy of these technologies restrict their application in primary cells. This article describes a protocol for penetrant, reproducible, and fast CRISPR-Cas9 genome editing in cell cultures derived from primary cells. The protocol employs transient nucleofection of ribonucleoprotein complexes composed of chemically synthesized 2'-O-methyl-3'phosphorothioate-modified single guide RNAs (sgRNAs) and purified Cas9 protein. It can be used both for targeted insertion-deletion mutation (indel) formation at up to >90% efficiency (via use of a single sgRNA) and for targeted deletion of genomic regions (via combined use of multiple sgRNAs). This article provides examples of the nucleofection buffer and programs that are optimal for patient-derived glioblastoma (GBM) stem-like cells (GSCs) and human neural stem/progenitor cells (NSCs), but the protocol can be readily applied to other primary cell cultures by modifying the nucleofection conditions. In summary, this is a relatively simple method that can be used for highly efficient and fast gene knockout, as well as for targeted genomic deletions, even in hyperdiploid cells such as many cancer stem-like cells. © 2020 Wiley Periodicals LLC Basic Protocol: Cas9:sgRNA ribonucleoprotein nucleofection for insertion-deletion (indel) mutation and genomic deletion generation in primary cell cultures.