Genomic Copy Number Dictates a Gene-Independent Cell Response to CRISPR/Cas9 Targeting

Aguirre, Andrew J.; Meyers, Robin M.; Weir, Barbara A.; Vázquez, Francisca; Chen, Ken; Ben‐David, Uri; Cook, April; Ha, Gavin; Harrington, William F.; Doshi, Mihir B.; Kost‐Alimova, Maria; Gill, Stanley; Han, Xu; Ali, Levi D.; Jiang, Guozhi; Pantel, Sasha; Lee, Yenarae; Goodale, Amy; Cherniack, Andrew D.; Oh, Coyin; Kryukov, Gregory V.; Cowley, Glenn S.; Garraway, Levi A.; Stegmaier, Kimberly; Roberts, Charles W.M.; Golub, Todd R.; Meyerson, Matthew; Root, David E.; Tsherniak, Aviad; Hahn, William C.

doi:10.1158/2159-8290.cd-16-0154

Cited by 511 publications

(614 citation statements)

References 48 publications

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“…1D). Furthermore, we investigated the gene copy number effects on CRISPR screen performance and confirmed that genes in genomic amplified regions tend to exhibit more negative selection as revealed by recent studies (16,26) (Fig. S1D and Dataset S1).…”

Section: Resultssupporting

confidence: 74%

Genome-wide CRISPR screen identifies HNRNPL as a prostate cancer dependency regulating RNA splicing

Teng

Chen

Xiao

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

274

244

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Alternative RNA splicing plays an important role in cancer. To determine which factors involved in RNA processing are essential in prostate cancer, we performed a genome-wide CRISPR/Cas9 knockout screen to identify the genes that are required for prostate cancer growth. Functional annotation defined a set of essential spliceosome and RNA binding protein (RBP) genes, including most notably heterogeneous nuclear ribonucleoprotein L (HNRNPL). We defined the HNRNPL-bound RNA landscape by RNA immunoprecipitation coupled with next-generation sequencing and linked these RBP-RNA interactions to changes in RNA processing. HNRNPL directly regulates the alternative splicing of a set of RNAs, including those encoding the androgen receptor, the key lineage-specific prostate cancer oncogene. HNRNPL also regulates circular RNA formation via back splicing. Importantly, both HNRNPL and its RNA targets are aberrantly expressed in human prostate tumors, supporting their clinical relevance. Collectively, our data reveal HNRNPL and its RNA clients as players in prostate cancer growth and potential therapeutic targets.CRISPR screen | HNRNPL | prostate cancer | RNA binding protein | alternative splicing P rostate cancer is among the most prevalent adult malignancies in developed countries. The principal treatment for prostate cancer once it is no longer amenable to surgery or radiation treatment is androgen deprivation therapy, which targets androgen or androgen receptor (AR) signaling. However, resistance to androgen deprivation therapy often develops and leads to a state termed "castration-resistant prostate cancer," which still lacks an effective cure (1-3). Therefore, significant efforts have been devoted to better understand the mechanism of oncogenesis and to develop additional effective therapeutics targeting pivotal oncogenes, cancer-related signal transduction pathways, and epigenetic regulators (4, 5).Alternative RNA splicing is a fundamental cellular process by which a single gene can give rise to multiple different transcripts and proteins. This process is tightly regulated by core spliceosomes and other splicing factors, such as the serine/ arginine-rich family of proteins and heterogeneous nuclear ribonucleoproteins (hnRNPs) (6, 7). Multiple studies indicate that deregulation of alternative splicing is implicated in cancer progression and that the splicing machinery may be targeted therapeutically (8-10). In addition to RNA splicing, the physical interactions between RNAs and RNA binding proteins (RBPs) underlie multiple RNA processing steps, such as capping, polyadenylation, transport, localization, modification, and translation, thereby regulating many aspects of RNA fate (11).Which RBPs and their related RNA processing steps are functionally important, especially in prostate cancer, remains elusive.The recent implementation of the clustered regularly interspaced short palindromic repeats (CRISPR)-associated nuclease Cas9 genome editing system has proved effective in high-throughput lossof-function screens (12)(13)(14)...

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Section: Resultssupporting

confidence: 74%

Genome-wide CRISPR screen identifies HNRNPL as a prostate cancer dependency regulating RNA splicing

Teng

Chen

Xiao

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

274

244

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“…In aneuploid cancer cell models, the genes located in amplified regions scored as lethal, indicating false positive results as already described by Aguirre et al (32). Thus, the number of loci representing the gene of interest in a cancer cell line under investigation is of crucial importance for the physiological response to CRISPR/Cas9 and for the experimental interpretation.…”

Section: Discussionmentioning

confidence: 71%

“…Two recent studies demonstrated that CRISPR/Cas9 is not suitable in cellular cancer models showing gene amplifications, as overrepresented genomic regions cause false positive results. Aguirre et al (32) provided evidence that DNA breaks generated by the CRISPR/Cas9 system lead to gene-independent cell toxicity and the number of DNA cuts rather than the gene knockout predicts the cellular response.…”

Section: Discussionmentioning

confidence: 99%

Dynamics of CRISPR/Cas9-mediated genomic editing of the AXL locus in hepatocellular carcinoma cells

et al. 2017

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Abstract. Genomic editing using the CRISPR/Cas9 technology allows selective interference with gene expression. With this method, a multitude of haploid and diploid cells from different organisms have been employed to successfully generate knockouts of genes coding for proteins or small RNAs. Yet, cancer cells exhibiting an aberrant ploidy are considered to be less accessible to CRISPR/Cas9-mediated genomic editing, as amplifications of the targeted gene locus could hamper its effectiveness. Here we examined the suitability of CRISPR/Cas9 to knockout the receptor tyrosine kinase Axl in the human hepatoma cell lines HLF and SNU449. The genomic editing events were validated in two single cell clones each from putative HLF and SNU449 knockout cells (HLF-Axl --1, HLF-Axl --2, SNU449-Axl --1, SNU449-Axl --2). Sequence analysis of respective AXL loci revealed one to six editing events in each individual Axl -clone. The majority of insertions and deletions in the AXL gene at exon 7/8 resulted in a frameshift and thus a premature stop in the coding region.However, one genomic editing event led to an insertion of two amino acids resulting in an altered protein sequence rather than in a frameshift in the AXL locus of the SNU449-Axl --1 cells. Notably, while no Axl protein expression could be detected by immunoblotting in all four cell clones, both expression of total Axl as well as release of soluble Axl into the supernatant was observed by ELISA in incompletely edited SNU449-Axl --1 cells. Importantly, a comparative genomic hybridization array revealed comparable genomic changes in Axl knockout cells as well as in cells expressing Cas9 nickase without guide RNAs in SNU449 and HLF cells, indicating vast alterations in genomic DNA triggered by nickase. Together, these data show that the dynamics of CRISPR/Cas9 may cause incomplete editing events in cancer cell lines, as gene copy numbers vary based on genomic heterogeneity.

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“…Multiple approaches fied. Cell lines stably expressing Cas9 were generated and Cas9 activity confirmed as previously described (26). Stable cell lines were then transduced with the Avana library and passaged in triplicate for 21 days, and the relative enrichment or depletion of guides was assessed using massively parallel sequencing and compared with the initial plasmid pool.…”

Section: Crispr-cas9 Screen Reveals a Mycn-amplified Neuroblastoma Dementioning

confidence: 99%

“…Stable cell lines were then transduced with the Avana library and passaged in triplicate for 21 days, and the relative enrichment or depletion of guides was assessed using massively parallel sequencing and compared with the initial plasmid pool. Data were normalized across cell lines, and a gene-level dependency score was calculated using the recently described CERES algorithm that takes into account the increased "cutting toxicity" observed in regions of copy number gain (24,26). We then identified preferential genetic dependencies in neuroblastoma cell lines by applying independent component analysis (ICA) (27,28) to the CRISPR-Cas9 data.…”

Section: Crispr-cas9 Screen Reveals a Mycn-amplified Neuroblastoma Dementioning

confidence: 99%

CRISPR-Cas9 screen reveals a MYCN-amplified neuroblastoma dependency on EZH2

Chen

Alexe

Dharia

et al. 2017

Journal of Clinical Investigation

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Genomic Copy Number Dictates a Gene-Independent Cell Response to CRISPR/Cas9 Targeting

Cited by 511 publications

References 48 publications

Genome-wide CRISPR screen identifies HNRNPL as a prostate cancer dependency regulating RNA splicing

Genome-wide CRISPR screen identifies HNRNPL as a prostate cancer dependency regulating RNA splicing

Dynamics of CRISPR/Cas9-mediated genomic editing of the AXL locus in hepatocellular carcinoma cells

CRISPR-Cas9 screen reveals a MYCN-amplified neuroblastoma dependency on EZH2

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