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Article ; Online: Generating Single Cell-Derived Knockout Clones in Mammalian Cells with CRISPR/Cas9.

Giuliano, Christopher J / Lin, Ann / Girish, Vishruth / Sheltzer, Jason M

2019 Volume 128, Issue 1, Page(s) e100

Abstract: 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. ... ...

Abstract	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.
MeSH term(s)	Animals ; CRISPR-Cas Systems ; Cell Line ; Clone Cells ; Gene Knockout Techniques/methods ; HEK293 Cells ; Humans ; Mammals ; Plasmids ; RNA, Guide, CRISPR-Cas Systems ; Transfection
Chemical Substances	RNA, Guide, CRISPR-Cas Systems
Language	English
Publishing date	2019-09-03
Publishing country	United States
Document type	Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't ; Research Support, U.S. Gov't, Non-P.H.S.
ISSN	1934-3647
ISSN (online)	1934-3647
DOI	10.1002/cpmb.100
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Article: Functional profiling of the

Giuliano, Christopher J / Wei, Kenneth J / Harling, Faye M / Waldman, Benjamin S / Farringer, Madeline A / Boydston, Elizabeth A / Lan, Tammy C T / Thomas, Raina W / Herneisen, Alice L / Sanderlin, Allen G / Coppens, Isabelle / Dvorin, Jeffrey D / Lourido, Sebastian

bioRxiv : the preprint server for biology

2023

Abstract: Within a host, pathogens encounter a diverse and changing landscape of cell types, nutrients, and immune responses. Examining host-pathogen interactions in animal models can therefore reveal aspects of infection absent from cell culture. We use CRISPR- ... ...

Abstract	Within a host, pathogens encounter a diverse and changing landscape of cell types, nutrients, and immune responses. Examining host-pathogen interactions in animal models can therefore reveal aspects of infection absent from cell culture. We use CRISPR-based screens to functionally profile the entire genome of the model apicomplexan parasite
Language	English
Publishing date	2023-03-06
Publishing country	United States
Document type	Preprint
DOI	10.1101/2023.03.05.531216
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Article ; Online: A positive feedback loop controls Toxoplasma chronic differentiation.

Licon, M Haley / Giuliano, Christopher J / Chan, Alex W / Chakladar, Sundeep / Eberhard, Julia N / Shallberg, Lindsey A / Chandrasekaran, Sambamurthy / Waldman, Benjamin S / Koshy, Anita A / Hunter, Christopher A / Lourido, Sebastian

Nature microbiology

2023 Volume 8, Issue 5, Page(s) 889–904

Abstract: Successful infection strategies must balance pathogen amplification and persistence. In the obligate intracellular parasite Toxoplasma gondii this is accomplished through differentiation into dedicated cyst-forming chronic stages that avoid clearance by ... ...

Abstract	Successful infection strategies must balance pathogen amplification and persistence. In the obligate intracellular parasite Toxoplasma gondii this is accomplished through differentiation into dedicated cyst-forming chronic stages that avoid clearance by the host immune system. The transcription factor BFD1 is both necessary and sufficient for stage conversion; however, its regulation is not understood. In this study we examine five factors that are transcriptionally activated by BFD1. One of these is a cytosolic RNA-binding protein of the CCCH-type zinc-finger family, which we name bradyzoite formation deficient 2 (BFD2). Parasites lacking BFD2 fail to induce BFD1 and are consequently unable to fully differentiate in culture or in mice. BFD2 interacts with the BFD1 transcript under stress, and deletion of BFD2 reduces BFD1 protein levels but not messenger RNA abundance. The reciprocal effects on BFD2 transcription and BFD1 translation outline a positive feedback loop that enforces the chronic-stage gene-expression programme. Thus, our findings help explain how parasites both initiate and commit to chronic differentiation. This work provides new mechanistic insight into the regulation of T. gondii persistence, and can be exploited in the design of strategies to prevent and treat these key reservoirs of human infection.
MeSH term(s)	Mice ; Animals ; Humans ; Toxoplasma/metabolism ; Feedback ; Gene Expression Regulation ; Transcription Factors/genetics
Chemical Substances	Transcription Factors
Language	English
Publishing date	2023-04-20
Publishing country	England
Document type	Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't
ISSN	2058-5276
ISSN (online)	2058-5276
DOI	10.1038/s41564-023-01358-2
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Article ; Online: Author Correction: A positive feedback loop controls Toxoplasma chronic differentiation.

Nature microbiology

2023 Volume 9, Issue 4, Page(s) 1145

Language	English
Publishing date	2023-08-14
Publishing country	England
Document type	Published Erratum
ISSN	2058-5276
ISSN (online)	2058-5276
DOI	10.1038/s41564-023-01467-y
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Article ; Online: CRISPR/Cas9 mutagenesis invalidates a putative cancer dependency targeted in on-going clinical trials.

Lin, Ann / Giuliano, Christopher J / Sayles, Nicole M / Sheltzer, Jason M

eLife

2017 Volume 6

Abstract: The Maternal Embryonic Leucine Zipper Kinase (MELK) has been reported to be a genetic dependency in several cancer types. MELK RNAi and small-molecule inhibitors of MELK block the proliferation of various cancer cell lines, and MELK knockdown has been ... ...

Abstract	The Maternal Embryonic Leucine Zipper Kinase (MELK) has been reported to be a genetic dependency in several cancer types. MELK RNAi and small-molecule inhibitors of MELK block the proliferation of various cancer cell lines, and MELK knockdown has been described as particularly effective against the highly-aggressive basal/triple-negative subtype of breast cancer. Based on these preclinical results, the MELK inhibitor OTS167 is currently being tested as a novel chemotherapy agent in several clinical trials. Here, we report that mutagenizing MELK with CRISPR/Cas9 has no effect on the fitness of basal breast cancer cell lines or cell lines from six other cancer types. Cells that harbor null mutations in MELK exhibit wild-type doubling times, cytokinesis, and anchorage-independent growth. Furthermore, MELK-knockout lines remain sensitive to OTS167, suggesting that this drug blocks cell division through an off-target mechanism. In total, our results undermine the rationale for a series of current clinical trials and provide an experimental approach for the use of CRISPR/Cas9 in preclinical target validation that can be broadly applied.
Language	English
Publishing date	2017-03-24
Publishing country	England
Document type	Journal Article
ZDB-ID	2687154-3
ISSN	2050-084X ; 2050-084X
ISSN (online)	2050-084X
ISSN	2050-084X
DOI	10.7554/eLife.24179
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Article ; Online: MELK expression correlates with tumor mitotic activity but is not required for cancer growth.

Giuliano, Christopher J / Lin, Ann / Smith, Joan C / Palladino, Ann C / Sheltzer, Jason M

eLife

2018 Volume 7

Abstract: The Maternal Embryonic Leucine Zipper Kinase (MELK) has been identified as a promising therapeutic target in multiple cancer types. MELK over-expression is associated with aggressive disease, and MELK has been implicated in numerous cancer-related ... ...

Abstract	The Maternal Embryonic Leucine Zipper Kinase (MELK) has been identified as a promising therapeutic target in multiple cancer types. MELK over-expression is associated with aggressive disease, and MELK has been implicated in numerous cancer-related processes, including chemotherapy resistance, stem cell renewal, and tumor growth. Previously, we established that triple-negative breast cancer cell lines harboring CRISPR/Cas9-induced null mutations in MELK proliferate at wild-type levels in vitro (<xref ref-type="bibr" rid="bib34">Lin et al., 2017</xref>). Here, we generate several additional knockout clones of MELK and demonstrate that across cancer types, cells lacking MELK exhibit wild-type growth in vitro, under environmental stress, in the presence of cytotoxic chemotherapies, and in vivo. By combining our MELK-knockout clones with a recently described, highly specific MELK inhibitor, we further demonstrate that the acute inhibition of MELK results in no specific anti-proliferative phenotype. Analysis of gene expression data from cohorts of cancer patients identifies MELK expression as a correlate of tumor mitotic activity, explaining its association with poor clinical prognosis. In total, our results demonstrate the power of CRISPR/Cas9-based genetic approaches to investigate cancer drug targets, and call into question the rationale for treating patients with anti-MELK monotherapies.
MeSH term(s)	Animals ; Carcinogenesis ; Cell Line, Tumor ; Cell Proliferation ; Gene Expression ; Gene Knockout Techniques ; Humans ; Mice, Nude ; Neoplasms/pathology ; Protein-Serine-Threonine Kinases/metabolism
Chemical Substances	MELK protein, human (EC 2.7.1.-) ; Protein-Serine-Threonine Kinases (EC 2.7.11.1)
Language	English
Publishing date	2018-02-08
Publishing country	England
Document type	Journal Article ; Research Support, N.I.H., Extramural
ZDB-ID	2687154-3
ISSN	2050-084X ; 2050-084X
ISSN (online)	2050-084X
ISSN	2050-084X
DOI	10.7554/eLife.32838
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Article ; Online: Structure of the mitochondrial inner membrane AAA+ protease YME1 gives insight into substrate processing.

Puchades, Cristina / Rampello, Anthony J / Shin, Mia / Giuliano, Christopher J / Wiseman, R Luke / Glynn, Steven E / Lander, Gabriel C

Science (New York, N.Y.)

2017 Volume 358, Issue 6363

Abstract: We present an atomic model of a substrate-bound inner mitochondrial membrane AAA+ quality control protease in yeast, YME1. Our ~3.4-angstrom cryo-electron microscopy structure reveals how the adenosine triphosphatases (ATPases) form a closed spiral ... ...

Abstract	We present an atomic model of a substrate-bound inner mitochondrial membrane AAA+ quality control protease in yeast, YME1. Our ~3.4-angstrom cryo-electron microscopy structure reveals how the adenosine triphosphatases (ATPases) form a closed spiral staircase encircling an unfolded substrate, directing it toward the flat, symmetric protease ring. Three coexisting nucleotide states allosterically induce distinct positioning of tyrosines in the central channel, resulting in substrate engagement and translocation to the negatively charged proteolytic chamber. This tight coordination by a network of conserved residues defines a sequential, around-the-ring adenosine triphosphate hydrolysis cycle that results in stepwise substrate translocation. A hingelike linker accommodates the large-scale nucleotide-driven motions of the ATPase spiral relative to the planar proteolytic base. The translocation mechanism is likely conserved for other AAA+ ATPases.
MeSH term(s)	ATP-Dependent Proteases/chemistry ; ATP-Dependent Proteases/ultrastructure ; Adenosine Triphosphate/metabolism ; Cryoelectron Microscopy ; Hydrolysis ; Mitochondrial Membranes/enzymology ; Models, Molecular ; Protein Domains ; Protein Transport ; Saccharomyces cerevisiae/enzymology ; Saccharomyces cerevisiae Proteins/chemistry ; Saccharomyces cerevisiae Proteins/ultrastructure ; Substrate Specificity
Chemical Substances	Saccharomyces cerevisiae Proteins ; Adenosine Triphosphate (8L70Q75FXE) ; ATP-Dependent Proteases (EC 3.4.21.-) ; YME1 protein, S cerevisiae (EC 3.4.21.-)
Language	English
Publishing date	2017-11-02
Publishing country	United States
Document type	Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't
ZDB-ID	128410-1
ISSN	1095-9203 ; 0036-8075
ISSN (online)	1095-9203
ISSN	0036-8075
DOI	10.1126/science.aao0464
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Article ; Online: B-cell-specific checkpoint molecules that regulate anti-tumour immunity.

Bod, Lloyd / Kye, Yoon-Chul / Shi, Jingwen / Torlai Triglia, Elena / Schnell, Alexandra / Fessler, Johannes / Ostrowski, Stephen M / Von-Franque, Max Y / Kuchroo, Juhi R / Barilla, Rocky M / Zaghouani, Sarah / Christian, Elena / Delorey, Toni Marie / Mohib, Kanishka / Xiao, Sheng / Slingerland, Nadine / Giuliano, Christopher J / Ashenberg, Orr / Li, Zhaorong /

Rothstein, David M / Fisher, David E / Rozenblatt-Rosen, Orit / Sharpe, Arlene H / Quintana, Francisco J / Apetoh, Lionel / Regev, Aviv / Kuchroo, Vijay K

Nature

2023 Volume 619, Issue 7969, Page(s) 348–356

Abstract: The role of B cells in anti-tumour immunity is still debated and, accordingly, immunotherapies have focused on targeting T and natural killer cells to inhibit tumour ... ...

Abstract	The role of B cells in anti-tumour immunity is still debated and, accordingly, immunotherapies have focused on targeting T and natural killer cells to inhibit tumour growth
MeSH term(s)	Animals ; Mice ; B-Lymphocytes/cytology ; B-Lymphocytes/immunology ; B-Lymphocytes/metabolism ; Lymphocyte Activation ; Melanoma/immunology ; Melanoma/pathology ; Melanoma/prevention & control ; T-Lymphocytes/cytology ; T-Lymphocytes/immunology ; Flow Cytometry ; Melanoma, Experimental/immunology ; Melanoma, Experimental/pathology ; Lymph Nodes/cytology ; Lymph Nodes/immunology ; Antigen Presentation ; Receptors, Antigen, B-Cell/genetics ; Single-Cell Gene Expression Analysis ; Tumor Burden ; Interferon Type I
Chemical Substances	Havcr1 protein, mouse ; Lag3 protein, mouse ; T cell Ig and ITIM domain protein, mouse ; Receptors, Antigen, B-Cell ; Interferon Type I
Language	English
Publishing date	2023-06-21
Publishing country	England
Document type	Journal Article
ZDB-ID	120714-3
ISSN	1476-4687 ; 0028-0836
ISSN (online)	1476-4687
ISSN	0028-0836
DOI	10.1038/s41586-023-06231-0
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Article ; Online: Off-target toxicity is a common mechanism of action of cancer drugs undergoing clinical trials.

Lin, Ann / Giuliano, Christopher J / Palladino, Ann / John, Kristen M / Abramowicz, Connor / Yuan, Monet Lou / Sausville, Erin L / Lukow, Devon A / Liu, Luwei / Chait, Alexander R / Galluzzo, Zachary C / Tucker, Clara / Sheltzer, Jason M

Science translational medicine

2019 Volume 11, Issue 509

Abstract: Ninety-seven percent of drug-indication pairs that are tested in clinical trials in oncology never advance to receive U.S. Food and Drug Administration approval. While lack of efficacy and dose-limiting toxicities are the most common causes of trial ... ...

Abstract	Ninety-seven percent of drug-indication pairs that are tested in clinical trials in oncology never advance to receive U.S. Food and Drug Administration approval. While lack of efficacy and dose-limiting toxicities are the most common causes of trial failure, the reason(s) why so many new drugs encounter these problems is not well understood. Using CRISPR-Cas9 mutagenesis, we investigated a set of cancer drugs and drug targets in various stages of clinical testing. We show that-contrary to previous reports obtained predominantly with RNA interference and small-molecule inhibitors-the proteins ostensibly targeted by these drugs are nonessential for cancer cell proliferation. Moreover, the efficacy of each drug that we tested was unaffected by the loss of its putative target, indicating that these compounds kill cells via off-target effects. By applying a genetic target-deconvolution strategy, we found that the mischaracterized anticancer agent OTS964 is actually a potent inhibitor of the cyclin-dependent kinase CDK11 and that multiple cancer types are addicted to CDK11 expression. We suggest that stringent genetic validation of the mechanism of action of cancer drugs in the preclinical setting may decrease the number of therapies tested in human patients that fail to provide any clinical benefit.
MeSH term(s)	Antineoplastic Agents/toxicity ; CRISPR-Cas Systems/genetics ; Cell Line, Tumor ; Clinical Trials as Topic ; Clone Cells ; Cyclin-Dependent Kinases/antagonists & inhibitors ; Cyclin-Dependent Kinases/metabolism ; Drug Resistance, Neoplasm/drug effects ; Gene Knockout Techniques ; Genome, Human ; Humans ; Molecular Targeted Therapy ; Quinolones/pharmacology ; RNA Interference/drug effects ; Up-Regulation/drug effects
Chemical Substances	Antineoplastic Agents ; OTS964 ; Quinolones ; CDK11B protein, human (EC 2.7.11.22) ; Cyclin-Dependent Kinases (EC 2.7.11.22)
Language	English
Publishing date	2019-10-07
Publishing country	United States
Document type	Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't ; Research Support, U.S. Gov't, Non-P.H.S.
ZDB-ID	2518854-9
ISSN	1946-6242 ; 1946-6234
ISSN (online)	1946-6242
ISSN	1946-6234
DOI	10.1126/scitranslmed.aaw8412
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Article ; Online: FABP1 controls hepatic transport and biotransformation of Δ

Elmes, Matthew W / Prentis, Lauren E / McGoldrick, Luke L / Giuliano, Christopher J / Sweeney, Joseph M / Joseph, Olivia M / Che, Joyce / Carbonetti, Gregory S / Studholme, Keith / Deutsch, Dale G / Rizzo, Robert C / Glynn, Steven E / Kaczocha, Martin

Scientific reports

2019 Volume 9, Issue 1, Page(s) 7588

Abstract: The increasing use of medical marijuana highlights the importance of developing a better understanding of cannabinoid metabolism. Phytocannabinoids, including ∆ ...

Abstract	The increasing use of medical marijuana highlights the importance of developing a better understanding of cannabinoid metabolism. Phytocannabinoids, including ∆
MeSH term(s)	Animals ; Binding Sites ; Biotransformation ; Cells, Cultured ; Crystallography, X-Ray ; Dronabinol/administration & dosage ; Dronabinol/metabolism ; Fatty Acid-Binding Proteins/chemistry ; Fatty Acid-Binding Proteins/metabolism ; Female ; Hepatocytes/metabolism ; Liver/metabolism ; Male ; Mice, Inbred C57BL ; Models, Molecular
Chemical Substances	FABP1 protein, human ; Fabp1 protein, mouse ; Fatty Acid-Binding Proteins ; Dronabinol (7J8897W37S)
Language	English
Publishing date	2019-05-20
Publishing country	England
Document type	Journal Article ; Research Support, N.I.H., Extramural ; Research Support, U.S. Gov't, Non-P.H.S.
ZDB-ID	2615211-3
ISSN	2045-2322 ; 2045-2322
ISSN (online)	2045-2322
ISSN	2045-2322
DOI	10.1038/s41598-019-44108-3
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