Use of CRISPR-cas9 gene targeting for genome-scale CRISPR screening in a glioma stem-cell line

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• 作者：Dr Imran Noorani ; BA MBBChir^a ; ^b ; ^{in1@sanger.ac.uk" class="auth_mail" title="E-mail the corresponding author} ; Prof Allan Bradley ; FRS^a
• 刊名：The Lancet
• 出版年：2016
• 出版时间：25 February 2016
• 年：2016
• 卷：387
• 期：supp_S1
• 页码：S78
• 全文大小：50 K

文摘

The bacterial clustered regularly interspaced short palindromic repeats (CRISPR)–cas9 system has recently emerged as a highly efficient gene-editing technique, in which the enzyme cas9 cleaves a genetic target under guidance from a CRISPR guide RNA. Glioma stem-cells are a useful model for the study of glioblastomas, but whether CRISPR–cas9 can be efficiently applied in this model is unknown. We aimed to derive and test a single cas9-expressing clone from a glioma stem-cell line.

Methods

An established human glioblastoma stem-cell line, NCH421K, was stably cultured in neural stem-cell conditions as neurospheres. A cas9 vector was designed with piggybac repeats for genome integration, and a neomycin-resistance cassette. The cas9 vector and a piggybac transposase vector were transfected into the glioma stem cells and selected with G418. The neurospheres were then dissociated into single cells, and flow sorting was used to transfer one cell to each well of a 96-well plate.

Findings

With this approach, expression of cas9 RNA in the transfected glioma stem-cell line was over 100 times higher than in wild-type cells, as demonstrated by real-time PCR. Repeat testing showed that the level of cas9 expression did not decrease over 2 months, indicating stable expression. Between 2 and 4 weeks after single-cell dissociation, single clonal neurospheres were isolated. Expansion of three such clones revealed cas9 expression in all of them. Functional testing with green fluorescence protein (GFP) expression (revealed by flow cytometry) demonstrated that transfection with a CRISPR guide RNA against GFP caused complete loss of GFP expression in over 90% of cloned glioma stem cells.

Interpretation

Our results demonstrate that isolation of a single glioma stem-cell clone engineered to express cas9 can generate highly efficient gene knock-outs. This model will allow unprecedented functional genomic screens of glioma stem cells on a genome-wide scale to identify novel cancer genes.

Funding

Wellcome Trust Clinical Research Training Fellowship.