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USP7 CRISPR knock-out in T-ALL cells [Molt4_CRISPR_RNA-seq]

ABSTRACT: RNAseq analysis of USP7 CRISPR KO in T-ALL cell lines. Genetically modified T-ALL cells were generated using CRISPR-Cas9 technology. Briefly, the sgRNA was designed with at least 3bp of mismatch to any other site in the human genome to mitigate the risk of off-target editing. To generate the modified cells, 400,000 cells were transiently co-transfected with / without 0.5 ug pmaxGFP plasmid (Lonza), with 33 pmol spCas9 protein, 100 pmol chemically modified sgRNA (Synthego), and 100 pmol of blocking ssODN (IDT) via nucleofection (Lonza), using solutions and programs according to the manufacturer’s recommended protocol. Five days post-nucleofection, cells were single-cell sorted by FACs into 96-well plates. Cells were clonally expanded and verified the desired modification via targeted deep sequencing

ORGANISM(S): Homo sapiens

PROVIDER: GSE148523 | GEO | 2021/03/09

REPOSITORIES: GEO

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Project description: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.

Dataset Information

USP7 CRISPR knock-out in T-ALL cells [Molt4_CRISPR_RNA-seq]

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