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

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 (GBM) 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 distribution were measured via computational deconvolution of Sanger sequencing trace data. We found that this optimized technique routinely allows for >90% indel formation in only 3 days, without the need to create clonal lines for simple loss-of-function experiments. Using Western blotting, we observed near-total protein loss of target genes in cell pools. Additionally, we found that this approach allows for the creation of targeted genomic deletions. By using RNA-seq in edited NSCs to assess gene expression changes resulting from knockout of tumor suppressors commonly altered in GBM, we also demonstrated the utility of this method for quickly creating a series of gene knockouts that allow for the study of oncogenic activities. Conclusion: Our data suggest that this relatively simple method can be used for highly efficient and fast gene knockout, as well as for targeted genomic deletions, even in hyperdiploid cells (such as GSCs). This represents an extremely useful tool for the cancer research community when wishing to inactivate not only coding genes, but also non-coding RNAs, UTRs, enhancers, and promoters. This method can be readily applied to diverse cell types by varying the nucleofection conditions. To assess gene expression changes resulting from loss of tumor suppressors commonly altered in glioblastoma, we performed RNA sequencing on hTERT-transduced neural stem cells (NSC-CB660-TERT) harboring various successive knockouts of TP53, CDKN2A, PTEN, and NF1. Knockouts were created by transiently nucleofecting NSC-CB660-TERT cells with Cas9:sgRNA ribonucleoprotein complexes composed of purified Cas9 protein and chemically synthesized 2'-O-methyl 3’phosphorothioate-modified sgRNAs. Three different sgRNAs were used simultaneously for targeting each gene. The pools of various KO cells were sequenced, along with one cell clone harboring knockout of all 4 genes.