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Article ; Online: Application of CHyMErA Cas9-Cas12a combinatorial genome-editing platform for genetic interaction mapping and gene fragment deletion screening.

Aregger, Michael / Xing, Kun / Gonatopoulos-Pournatzis, Thomas

2021 Volume 16, Issue 10, Page(s) 4722–4765

Abstract: CRISPR-based forward genetic screening represents a powerful approach for the systematic characterization of gene function. Recent efforts have been directed toward establishing CRISPR-based tools for the programmable delivery of combinatorial genetic ... ...

Abstract	CRISPR-based forward genetic screening represents a powerful approach for the systematic characterization of gene function. Recent efforts have been directed toward establishing CRISPR-based tools for the programmable delivery of combinatorial genetic perturbations, most of which are mediated by a single nuclease and the expression of structurally identical guide backbones from two promoters. In contrast, we have developed CHyMErA (Cas hybrid for multiplexed editing and screening applications), which is based on the co-expression of Cas9 and Cas12a nucleases in conjunction with a hybrid guide RNA (hgRNA) engineered by the fusion of Cas9 and Cas12a guides and expressed from a single U6 promoter. CHyMErA is suitable for the high-throughput deletion of genetic segments including the excision of individual exons. Furthermore, CHyMErA enables the concomitant targeting of two or more genes and can thus be used for the systematic mapping of genetic interactions in mammalian cells. CHyMErA can also be applied for the perturbation of paralogous gene pairs, thereby allowing the capturing of phenotypic roles that would otherwise be masked because of genetic redundancy. Here, we provide instructions for the cloning of hgRNA screening libraries and individual hgRNA constructs and offer guidelines for designing and performing combinatorial pooled genetic screens using CHyMErA. Starting with the generation of Cas9- and Cas12a-expressing cell lines, CHyMErA screening can be implemented within 15-20 weeks.
MeSH term(s)	CRISPR-Cas Systems ; Cell Line ; Gene Deletion ; Gene Editing ; Humans
Language	English
Publishing date	2021-09-10
Publishing country	England
Document type	Journal Article ; Research Support, Non-U.S. Gov't ; Review
ZDB-ID	2244966-8
ISSN	1750-2799 ; 1754-2189
ISSN (online)	1750-2799
ISSN	1754-2189
DOI	10.1038/s41596-021-00595-1
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Article ; Online: Reproducibility metrics for context-specific CRISPR screens.

Billmann, Maximilian / Ward, Henry N / Aregger, Michael / Costanzo, Michael / Andrews, Brenda J / Boone, Charles / Moffat, Jason / Myers, Chad L

Cell systems

2023 Volume 14, Issue 5, Page(s) 418–422.e2

Abstract: CRISPR screens are used extensively to systematically interrogate the phenotype-to-genotype problem. In contrast to early CRISPR screens, which defined core cell fitness genes, most current efforts now aim to identify context-specific phenotypes that ... ...

Abstract	CRISPR screens are used extensively to systematically interrogate the phenotype-to-genotype problem. In contrast to early CRISPR screens, which defined core cell fitness genes, most current efforts now aim to identify context-specific phenotypes that differentiate a cell line, genetic background, or condition of interest, such as a drug treatment. While CRISPR-related technologies have shown great promise and a fast pace of innovation, a better understanding of standards and methods for quality assessment of CRISPR screen results is crucial to guide technology development and application. Specifically, many commonly used metrics for quantifying screen quality do not accurately measure the reproducibility of context-specific hits. We highlight the importance of reporting reproducibility statistics that directly relate to the purpose of the screen and suggest the use of metrics that are sensitive to context-specific signal. A record of this paper's transparent peer review process is included in the supplemental information.
MeSH term(s)	Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Reproducibility of Results ; Phenotype ; Cell Line
Language	English
Publishing date	2023-05-16
Publishing country	United States
Document type	Journal Article ; Research Support, U.S. Gov't, Non-P.H.S. ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't
ZDB-ID	2854138-8
ISSN	2405-4720 ; 2405-4712
ISSN (online)	2405-4720
ISSN	2405-4712
DOI	10.1016/j.cels.2023.04.003
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Article ; Online: Regulation of mRNA capping in the cell cycle.

Aregger, Michael / Cowling, Victoria H

RNA biology

2016 Volume 14, Issue 1, Page(s) 11–14

Abstract: The mRNA cap structure, which is added to nascent RNA pol II transcripts, recruits the protein complexes required for pre-mRNA transcript processing, mRNA export and translation initiation. The enzymes which catalyze mRNA cap synthesis are regulated by ... ...

Abstract	The mRNA cap structure, which is added to nascent RNA pol II transcripts, recruits the protein complexes required for pre-mRNA transcript processing, mRNA export and translation initiation. The enzymes which catalyze mRNA cap synthesis are regulated by cellular signaling pathways which impact on their expression, localization and activity. Here we discuss the recent observation that the mRNA cap methyltransferase, RNMT, is phosphorylated on Thr-77 by CDK1-cyclin B1, which regulates its activity and the proteins with which it interacts. RNMT Thr-77 phosphorylation provides a burst of mRNA cap methyltransferase activity during early G1 phase at a time when transcription is reactivated following completion of the cell cycle. This co-ordination of transcription and mRNA capping makes an important contribution to gene expression in the cell; preventing RNMT Thr-77 phosphorylation inhibits cell proliferation. Here we discuss these findings and how mRNA cap synthesis may be regulated in other scenarios.
MeSH term(s)	Animals ; Cell Cycle/genetics ; Gene Expression Regulation ; Humans ; Methylation ; Methyltransferases/metabolism ; Phosphorylation ; Protein Biosynthesis ; RNA Caps/metabolism ; RNA, Messenger/genetics ; RNA, Messenger/metabolism ; Transcription, Genetic
Chemical Substances	RNA Caps ; RNA, Messenger ; Methyltransferases (EC 2.1.1.-) ; mRNA (guanine(N7))-methyltransferase (EC 2.1.1.56)
Language	English
Publishing date	2016-10-28
Publishing country	United States
Document type	Journal Article ; Review ; Research Support, Non-U.S. Gov't
ISSN	1555-8584
ISSN (online)	1555-8584
DOI	10.1080/15476286.2016.1251540
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Article ; Online: A Method to Map Gene Essentiality of Human Pluripotent Stem Cells by Genome-Scale CRISPR Screens with Inducible Cas9.

Mair, Barbara / Aregger, Michael / Tong, Amy H Y / Chan, Katherine S K / Moffat, Jason

Methods in molecular biology (Clifton, N.J.)

2021 Volume 2377, Page(s) 1–27

Abstract: Human pluripotent stem cells (hPSCs) have the capacity for self-renewal and differentiation into most cell types and, in contrast to widely used cell lines, are karyotypically normal and non-transformed. Hence, hPSCs are considered the gold-standard ... ...

Abstract	Human pluripotent stem cells (hPSCs) have the capacity for self-renewal and differentiation into most cell types and, in contrast to widely used cell lines, are karyotypically normal and non-transformed. Hence, hPSCs are considered the gold-standard system for modelling diseases, especially in the field of regenerative medicine. Despite widespread research use of hPSCs and induced pluripotent stem cells (iPSCs), the systematic understanding of pluripotency and lineage differentiation mechanisms are still incomplete. Before tackling the complexities of lineage differentiation with genetic screens, it is critical to catalogue the general genetic requirements for cell fitness and proliferation in the pluripotent state and assess their plasticity under commonly used culture conditions.We describe a method to map essential genetic determinants of hPSC fitness and pluripotency, herein defined as cell reproduction, by genome-scale loss-of-function CRISPR screens in an inducible S. pyogenes Cas9 H1 hPSC line. To address questions of context-dependent gene essentiality, we include protocols for screening hPSCs cultured on feeder cells and laminin, two commonly used growth substrates. This method establishes parameters for genome-wide screens in hPSCs, making human stem cells amenable for functional genomics approaches to facilitate investigation of hPSC biology.
MeSH term(s)	CRISPR-Cas Systems/genetics ; Cell Differentiation/genetics ; Feeder Cells ; Genes, Essential ; Humans ; Pluripotent Stem Cells
Language	English
Publishing date	2021-10-28
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ISSN	1940-6029
ISSN (online)	1940-6029
DOI	10.1007/978-1-0716-1720-5_1
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Article ; Online: Abrogation of the G2/M checkpoint as a chemo sensitization approach for alkylating agents.

Lang, Fengchao / Cornwell, James A / Kaur, Karambir / Elmogazy, Omar / Zhang, Wei / Zhang, Meili / Song, Hua / Sun, Zhonghe / Wu, Xiaolin / Aladjem, Mirit I / Aregger, Michael / Cappell, Steven D / Yang, Chunzhang

Neuro-oncology

2023

Abstract: Background: The cell cycle is tightly regulated by checkpoints, playing a vital role in controlling its progression and timing. Cancer cells exploit the G2/M checkpoint, which serves as a resistance mechanism against genotoxic anti-cancer treatments, ... ...

Abstract	Background: The cell cycle is tightly regulated by checkpoints, playing a vital role in controlling its progression and timing. Cancer cells exploit the G2/M checkpoint, which serves as a resistance mechanism against genotoxic anti-cancer treatments, allowing for DNA repair prior to cell division. Manipulating cell cycle timing has emerged as a potential strategy to augment the effectiveness of DNA damage-based therapies. Methods: In this study, we conducted a forward genome wide CRISPR/Cas9 screening with repeated exposure to the alkylating agent temozolomide (TMZ) to investigate the mechanisms underlying tumor cell survival under genotoxic stress. Results: Our findings revealed that canonical DNA repair pathways, including ATM/Fanconi and mismatch repair, determine cell fate under genotoxic stress. Notably, we identified the critical role of PKMYT1, in ensuring cell survival. Depletion of PKMYT1 led to overwhelming TMZ-induced cytotoxicity in cancer cells. Isobologram analysis demonstrated potent drug synergy between alkylating agents and a Myt1 kinase inhibitor, RP-6306. Mechanistically, inhibiting Myt1 forced G2/M-arrested cells into an unscheduled transition to the mitotic phase without complete resolution of DNA damage. This forced entry into mitosis, along with persistent DNA damage, resulted in severe mitotic abnormalities. Ultimately, these aberrations led to mitotic exit with substantial apoptosis. Preclinical animal studies demonstrated that the combination regimen involving TMZ and RP-6306 prolonged the overall survival of glioma-bearing mice. Conclusion: Collectively, our findings highlight the potential of targeting cell cycle timing through Myt1 inhibition as an effective strategy to enhance the efficacy of current standard cancer therapies, potentially leading to improved disease outcomes.
Language	English
Publishing date	2023-12-22
Publishing country	England
Document type	Journal Article
ZDB-ID	2028601-6
ISSN	1523-5866 ; 1522-8517
ISSN (online)	1523-5866
ISSN	1522-8517
DOI	10.1093/neuonc/noad252
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Zs.A 5307: Show issues

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Article ; Online: Human cap methyltransferase (RNMT) N-terminal non-catalytic domain mediates recruitment to transcription initiation sites.

Aregger, Michael / Cowling, Victoria H

The Biochemical journal

2013 Volume 455, Issue 1, Page(s) 67–73

Abstract: Gene expression in eukaryotes is dependent on the mRNA methyl cap which mediates mRNA processing and translation initiation. Synthesis of the methyl cap initiates with the addition of 7-methylguanosine to the initiating nucleotide of RNA pol II ( ... ...

Abstract	Gene expression in eukaryotes is dependent on the mRNA methyl cap which mediates mRNA processing and translation initiation. Synthesis of the methyl cap initiates with the addition of 7-methylguanosine to the initiating nucleotide of RNA pol II (polymerase II) transcripts, which occurs predominantly during transcription and in mammals is catalysed by RNGTT (RNA guanylyltransferase and 5' phosphatase) and RNMT (RNA guanine-7 methyltransferase). RNMT has a methyltransferase domain and an N-terminal domain whose function is unclear; it is conserved in mammals, but not required for cap methyltransferase activity. In the present study we report that the N-terminal domain is necessary and sufficient for RNMT recruitment to transcription initiation sites and that recruitment occurs in a DRB (5,6-dichloro-1-β-D-ribofuranosylbenzimidazole)-dependent manner. The RNMT-activating subunit, RAM (RNMT-activating miniprotein), is also recruited to transcription initiation sites via an interaction with RNMT. The RNMT N-terminal domain is required for transcript expression, translation and cell proliferation.
MeSH term(s)	Binding Sites ; Cell Line, Tumor ; Cell Proliferation/drug effects ; Dichlororibofuranosylbenzimidazole/pharmacology ; Enzyme Inhibitors/pharmacology ; Gene Expression Regulation/drug effects ; Humans ; Methyltransferases/genetics ; Methyltransferases/metabolism ; Peptide Chain Initiation, Translational/drug effects ; Protein Binding ; Protein Structure, Tertiary ; RNA-Binding Proteins/genetics ; RNA-Binding Proteins/metabolism ; Signal Transduction ; Transcription Initiation Site ; Transcription, Genetic/drug effects
Chemical Substances	Enzyme Inhibitors ; RNA-Binding Proteins ; Dichlororibofuranosylbenzimidazole (53-85-0) ; Methyltransferases (EC 2.1.1.-) ; RAMAC protein, human (EC 2.1.1.-) ; mRNA (guanine(N7))-methyltransferase (EC 2.1.1.56)
Language	English
Publishing date	2013-07-17
Publishing country	England
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	2969-5
ISSN	1470-8728 ; 0006-2936 ; 0306-3275 ; 0264-6021
ISSN (online)	1470-8728
ISSN	0006-2936 ; 0306-3275 ; 0264-6021
DOI	10.1042/BJ20130378
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Article ; Online: E2F1-dependent methyl cap formation requires RNA pol II phosphorylation.

Aregger, Michael / Cowling, Victoria H

Cell cycle (Georgetown, Tex.)

2012 Volume 11, Issue 11, Page(s) 2146–2148

Abstract: Gene expression is a process integral to cell proliferation. The E2F family of transcription factors upregulates expression of transcripts whose products are essential for cell cycle progression. Here, we report that E2F1 promotes gene expression by an ... ...

Abstract	Gene expression is a process integral to cell proliferation. The E2F family of transcription factors upregulates expression of transcripts whose products are essential for cell cycle progression. Here, we report that E2F1 promotes gene expression by an additional mechanism, that is, formation of the methyl cap on RNA pol II transcripts. The methyl cap is required for mRNA maturation, expression and stability. We demonstrate that E2F1 increases RNA pol II phosphorylation, which promotes recruitment of the methyl cap synthetic enzymes. Upregulation of RNA pol II phosphorylation is required for E2F1-dependent methyl cap formation.
MeSH term(s)	Animals ; CDC2 Protein Kinase/metabolism ; Cells, Cultured ; DNA Methylation ; E2F1 Transcription Factor/metabolism ; Gene Expression ; Phosphorylation ; RNA Polymerase II/metabolism ; Rats
Chemical Substances	E2F1 Transcription Factor ; CDC2 Protein Kinase (EC 2.7.11.22) ; RNA Polymerase II (EC 2.7.7.-)
Language	English
Publishing date	2012-06-01
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	2146183-1
ISSN	1551-4005 ; 1538-4101 ; 1554-8627
ISSN (online)	1551-4005
ISSN	1538-4101 ; 1554-8627
DOI	10.4161/cc.20620
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Article ; Online: Extensive mapping of an innate immune network with CRISPR.

Aregger, Michael / Hart, Traver / Moffat, Jason

Molecular systems biology

2015 Volume 11, Issue 7, Page(s) 821

Abstract: The application of the CRISPR‐Cas9 system marks a major breakthrough for genetic screens, particularly in mammalian cells where high‐throughput targeted gene editing has been lacking. Parnas et al (2015) apply this screening technology to mouse bone ... ...

Abstract	The application of the CRISPR‐Cas9 system marks a major breakthrough for genetic screens, particularly in mammalian cells where high‐throughput targeted gene editing has been lacking. Parnas et al (2015) apply this screening technology to mouse bone marrow‐derived dendritic cells in order to study the regulation of the immune response triggered by PAMPs. Through integrated analysis of gene knockouts in conjunction with changes in protein and mRNA expression, CRISPR screens are facilitating dissection of immune regulatory networks at unprecedented resolution.
MeSH term(s)	Animals ; Bacterial Proteins/genetics ; CRISPR-Cas Systems ; Dendritic Cells/immunology ; Gene Knockdown Techniques ; Gene Regulatory Networks ; Immunity, Innate ; Mice ; RNA Editing
Chemical Substances	Bacterial Proteins
Language	English
Publishing date	2015-07-24
Publishing country	England
Document type	Journal Article
ISSN	1744-4292
ISSN (online)	1744-4292
DOI	10.15252/msb.20156373
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Article ; Online: Pooled Lentiviral CRISPR-Cas9 Screens for Functional Genomics in Mammalian Cells.

Aregger, Michael / Chandrashekhar, Megha / Tong, Amy Hin Yan / Chan, Katherine / Moffat, Jason

Methods in molecular biology (Clifton, N.J.)

2018 Volume 1869, Page(s) 169–188

Abstract: CRISPR-Cas9 technology provides a simple way to introduce targeted mutations into mammalian cells to induce loss-of-function phenotypes. The CRISPR-Cas9 system has now successfully been applied for genetic screens in many cell types, providing a powerful ...

Abstract	CRISPR-Cas9 technology provides a simple way to introduce targeted mutations into mammalian cells to induce loss-of-function phenotypes. The CRISPR-Cas9 system has now successfully been applied for genetic screens in many cell types, providing a powerful tool for functional genomics with manifold applications. Genome-wide guide-RNA (gRNA) libraries allow facile generation of a pool of cells, each harboring a gene knockout mutation that can be used for the study of gene function, pathway analysis or the identification of genes required for cellular fitness. Furthermore, CRISPR genetic screens can be applied for the discovery of genes whose knockout sensitizes cells to drug treatments or mediates drug resistance. Here, we provide a detailed protocol discussing the necessary steps for the successful performance of pooled CRISPR-Cas9 screens.
MeSH term(s)	Animals ; CRISPR-Associated Protein 9/metabolism ; CRISPR-Cas Systems/genetics ; Cell Line ; Data Analysis ; Gene Knockout Techniques ; Gene Library ; Genetic Testing ; Genomics/methods ; High-Throughput Nucleotide Sequencing ; Lentivirus/genetics ; Mammals/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics
Chemical Substances	RNA, Guide, CRISPR-Cas Systems ; CRISPR-Associated Protein 9 (EC 3.1.-)
Language	English
Publishing date	2018-09-19
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ISSN	1940-6029
ISSN (online)	1940-6029
DOI	10.1007/978-1-4939-8805-1_15
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Article ; Online: Analysis of combinatorial CRISPR screens with the Orthrus scoring pipeline.

Ward, Henry N / Aregger, Michael / Gonatopoulos-Pournatzis, Thomas / Billmann, Maximilian / Ohsumi, Toshiro K / Brown, Kevin R / Blencowe, Benjamin J / Moffat, Jason / Myers, Chad L

Nature protocols

2021 Volume 16, Issue 10, Page(s) 4766–4798

Abstract: The continued improvement of combinatorial CRISPR screening platforms necessitates the development of new computational pipelines for scoring combinatorial screening data. Unlike for single-guide RNA (sgRNA) pooled screening platforms, combinatorial ... ...

Abstract	The continued improvement of combinatorial CRISPR screening platforms necessitates the development of new computational pipelines for scoring combinatorial screening data. Unlike for single-guide RNA (sgRNA) pooled screening platforms, combinatorial scoring for multiplexed systems is confounded by guide design parameters such as the number of gRNAs per construct, the position of gRNAs along constructs, and additional features that may impact gRNA expression, processing or capture. In this protocol we describe Orthrus, an R package for processing, scoring and analyzing combinatorial CRISPR screening data that addresses these challenges. This protocol walks through the application of Orthrus to previously published combinatorial screening data from the CHyMErA experimental system, a platform we recently developed that pairs Cas9 with Cas12a gRNAs and enables programmed targeting of multiple genomic sites. We demonstrate Orthrus' features for screen quality assessment and two distinct scoring modes for dual guide RNAs (dgRNAs) that target the same gene twice or dgRNAs that target two different genes. Running Orthrus requires basic R programming experience, ~5-10 min of computational time and 15-60 min total.
MeSH term(s)	CRISPR-Cas Systems ; Gene Editing ; RNA, Guide, CRISPR-Cas Systems
Chemical Substances	RNA, Guide, CRISPR-Cas Systems
Language	English
Publishing date	2021-09-10
Publishing country	England
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. ; Review
ZDB-ID	2244966-8
ISSN	1750-2799 ; 1754-2189
ISSN (online)	1750-2799
ISSN	1754-2189
DOI	10.1038/s41596-021-00596-0
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