CRISPR/Cas9 mutagenesis invalidates a putative cancer dependency targeted in on-going clinical trials

Lin, Andy; Giuliano, Christopher J.; Sayles, Nicole M.; Sheltzer, Jason M.

doi:10.7554/elife.24179

Cited by 118 publications

(132 citation statements)

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“…experiments to confirm knockout of the MELK kinase from cancer cell lines (Giuliano, Lin, Smith, Palladino, & Sheltzer, 2018;Lin, Giuliano, Sayles, & Sheltzer, 2017).…”

Section: Giuliano Et Almentioning

confidence: 99%

“…Rederiving a cell line from a single cell represents a significant genetic bottleneck, and experiments may be confounded if a single cellderived clone captures only a subset of the diversity present within the starting cell population. Indeed, some established cell lines are well known to exhibit significant interclonal heterogeneity; for instance, we and others have observed sizeable variation in proliferative capacity among clones derived from the widely used MDA-MB-231 breast cancer cell line (Giuliano et al, 2018;Khan, Kim, Shin, & Lee, 2017;Lin et al, 2017). Because of the possibility of parental heterogeneity, it is absolutely crucial that researchers analyze multiple independent knockout cell lines in order to ensure that any unexpected result is not a clonal artifact.…”

Section: Critical Parametersmentioning

confidence: 99%

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Generating Single Cell–Derived Knockout Clones in Mammalian Cells with CRISPR/Cas9

Giuliano

Lin

Girish

et al. 2019

CP Molecular Biology

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CRISPR/Cas9 technology enables the rapid generation of loss‐of‐function mutations in a targeted gene in mammalian cells. A single cell harboring those mutations can be used to establish a new cell line, thereby creating a CRISPR‐induced knockout clone. These clonal cell lines serve as crucial tools for exploring protein function, analyzing the consequences of gene loss, and investigating the specificity of biological reagents. However, the successful derivation of knockout clones can be technically challenging and may be complicated by multiple factors, including incomplete target ablation and interclonal heterogeneity. Here, we describe optimized protocols and plasmids for generating clonal knockouts in mammalian cell lines. We provide strategies for guide RNA design, CRISPR delivery, and knockout validation that facilitate the derivation of true knockout clones and are amenable to multiplexed gene targeting. These protocols will be broadly useful for researchers seeking to apply CRISPR to study gene function in mammalian cells. © 2019 The Authors.

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“…experiments to confirm knockout of the MELK kinase from cancer cell lines (Giuliano, Lin, Smith, Palladino, & Sheltzer, 2018;Lin, Giuliano, Sayles, & Sheltzer, 2017).…”

Section: Giuliano Et Almentioning

confidence: 99%

Section: Critical Parametersmentioning

confidence: 99%

Generating Single Cell–Derived Knockout Clones in Mammalian Cells with CRISPR/Cas9

Giuliano

Lin

Girish

et al. 2019

CP Molecular Biology

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“…Our analysis also showed that PAR-1 has a specific role in EMS cell division dynamics in a pathway separable from that of PIG-1. The mammalian homolog of PIG-1, MELK, is also nonessential for viability in mice ( (Lin et al, 2017;Wang et al, 2014), but is highly expressed in mammalian embryonic cells, hematopoietic cells and neural progenitor cells (Gil et al, 1997;Heyer et al, 1997;Nakano et al, 2005). MELK is likely to act redundantly with other mitotic kinases, which may include closely related MARK family kinases, homologs of PAR-1 (Drewes et al, 1997).…”

Section: Discussionmentioning

confidence: 99%

The kinases PIG-1 and PAR-1 act in redundant pathways to regulate asymmetric division in the EMS blastomere ofC. elegans

Liro

Morton

Rose

2017

Preprint

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The PAR-1 kinase of C. elegans is localized to the posterior of the one-cell embryo and its mutations affect asymmetric spindle placement and partitioning of cytoplasmic components in the first cell cycle. However, unlike mutations in the posteriorly localized PAR-2 protein, par-1 mutations do not cause failure to restrict the anterior PAR polarity complex. Further, it has been difficult to examine the role of PAR-1 in subsequent divisions due to the early defects in par-1 mutant embryos. Here we show that the PIG-1 kinase acts redundantly with PAR-1 to restrict the anterior PAR-3 protein for polarity maintenance in the one-cell embryo. By using a weak allele of par-1 that exhibits enhanced lethality when combined with a pig-1 mutation we have further explored roles for these genes in subsequent divisions. We find that both PIG-1 and PAR-1 regulate spindle orientation in the EMS blastomere of the four-cell stage embryo to ensure that it undergoes an asymmetric division. In this cell, PIG-1 and PAR-1 act in parallel pathways for spindle positioning, PIG-1 in the MES-1/SRC-1 pathway and PAR-1 in the Wnt pathway.

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“…Consequently, multiple drugs that inhibit MELK activity have been synthesized as a means for therapy . Although the MELK inhibitor OTS167 has been studied in several clinical trials (NCT01910545, NCT02768519, NCT02795520, and NCT02926690), Lin et al discovered that for TNBC, the mutagenesis of MELK with Cas9 did not affect the growth of TNBC cell lines . Furthermore, when CRISPR induced a null mutation at the reported drug target, there was no evident drug resistance.…”

Section: Crispr Technology For Breast Cancer Modelingmentioning

confidence: 99%

CRISPR Technology for Breast Cancer: Diagnostics, Modeling, and Therapy

Mintz

Gao

et al. 2018

Adv. Biosys.

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Molecularly, breast cancer represents a highly heterogenous family of neoplastic disorders, with substantial interpatient variations regarding genetic mutations, cell composition, transcriptional profiles, and treatment response. Consequently, there is an increasing demand for alternative diagnostic approaches aimed at the molecular annotation of the disease on a patient‐by‐patient basis and the design of more personalized treatments. The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR‐associated (Cas) technology enables the development of such novel approaches. For instance, in diagnostics, the use of the RNA‐specific C2c2 system allows ultrasensitive nucleic acid detection and could be used to characterize the mutational repertoire and transcriptional breast cancer signatures. In disease modeling, CRISPR/Cas9 technology can be applied to selectively engineer oncogenes and tumor‐suppressor genes involved in disease pathogenesis. In treatment, CRISPR/Cas9 can be used to develop gene‐therapy, while its catalytically‐dead variant (dCas9) can be applied to reprogram the epigenetic landscape of malignant cells. As immunotherapy becomes increasingly prominent in cancer treatment, CRISPR/Cas9 can engineer the immune cells to redirect them against cancer cells and potentiate antitumor immune responses. In this review, CRISPR strategies for the advancement of breast cancer diagnostics, modeling, and treatment are highlighted, culminating in a perspective on developing a precision medicine‐based approach against breast cancer.

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CRISPR/Cas9 mutagenesis invalidates a putative cancer dependency targeted in on-going clinical trials

Cited by 118 publications

References 58 publications

Generating Single Cell–Derived Knockout Clones in Mammalian Cells with CRISPR/Cas9

Generating Single Cell–Derived Knockout Clones in Mammalian Cells with CRISPR/Cas9

The kinases PIG-1 and PAR-1 act in redundant pathways to regulate asymmetric division in the EMS blastomere ofC. elegans

CRISPR Technology for Breast Cancer: Diagnostics, Modeling, and Therapy

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