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  1. Article: A CRISPR–dCas Toolbox for Genetic Engineering and Synthetic Biology

    Xu, Xiaoshu / Lei S. Qi

    Journal of molecular biology. 2019 Jan. 04, v. 431, no. 1

    2019  

    Abstract: Programmable control of gene expression is essential to understanding gene function, engineering cellular behaviors, and developing therapeutics. Beyond the gene editing applications enabled by the nuclease CRISPR–Cas9 and CRISPR–Cas12a, the invention of ...

    Abstract Programmable control of gene expression is essential to understanding gene function, engineering cellular behaviors, and developing therapeutics. Beyond the gene editing applications enabled by the nuclease CRISPR–Cas9 and CRISPR–Cas12a, the invention of the nuclease-dead Cas molecules (dCas9 and dCas12a) offers a platform for the precise control of genome function without gene editing. Diverse dCas tools have been developed, which constitute a comprehensive toolbox that allows for interrogation of gene function and modulation of the cellular behaviors. This review summarizes current applications of the dCas tools for transcription regulation, epigenetic engineering, genome imaging, genetic screens, and chromatin immunoprecipitation. We also highlight the advantages and existing challenges of the current dCas tools in genetic engineering and synthetic biology, and provide perspectives on future directions and applications.
    Keywords chromatin ; endodeoxyribonucleases ; gene editing ; genes ; image analysis ; precipitin tests ; synthetic biology ; therapeutics ; transcription (genetics)
    Language English
    Dates of publication 2019-0104
    Size p. 34-47.
    Publishing place Elsevier Ltd
    Document type Article
    ZDB-ID 80229-3
    ISSN 1089-8638 ; 0022-2836
    ISSN (online) 1089-8638
    ISSN 0022-2836
    DOI 10.1016/j.jmb.2018.06.037
    Database NAL-Catalogue (AGRICOLA)

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  2. Article ; Online: Enhanced Cas12a multi-gene regulation using a CRISPR array separator

    Jens P Magnusson / Antonio Ray Rios / Lingling Wu / Lei S Qi

    eLife, Vol

    2021  Volume 10

    Abstract: The type V-A Cas12a protein can process its CRISPR array, a feature useful for multiplexed gene editing and regulation. However, CRISPR arrays often exhibit unpredictable performance due to interference between multiple guide RNA (gRNAs). Here, we report ...

    Abstract The type V-A Cas12a protein can process its CRISPR array, a feature useful for multiplexed gene editing and regulation. However, CRISPR arrays often exhibit unpredictable performance due to interference between multiple guide RNA (gRNAs). Here, we report that Cas12a array performance is hypersensitive to the GC content of gRNA spacers, as high-GC spacers can impair activity of the downstream gRNA. We analyze naturally occurring CRISPR arrays and observe that natural repeats always contain an AT-rich fragment that separates gRNAs, which we term a CRISPR separator. Inspired by this observation, we design short, AT-rich synthetic separators (synSeparators) that successfully remove the disruptive effects between gRNAs. We further demonstrate enhanced simultaneous activation of seven endogenous genes in human cells using an array containing the synSeparator. These results elucidate a previously underexplored feature of natural CRISPR arrays and demonstrate how nature-inspired engineering solutions can improve multi-gene control in mammalian cells.
    Keywords CRISPR ; Cas12a ; multiplexed gene regulation ; CRISPR array ; separator ; GC content ; Medicine ; R ; Science ; Q ; Biology (General) ; QH301-705.5
    Subject code 612
    Language English
    Publishing date 2021-09-01T00:00:00Z
    Publisher eLife Sciences Publications Ltd
    Document type Article ; Online
    Database BASE - Bielefeld Academic Search Engine (life sciences selection)

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  3. Article ; Online: A comprehensive analysis and resource to use CRISPR-Cas13 for broad-spectrum targeting of RNA viruses

    Xueqiu Lin / Yanxia Liu / Augustine Chemparathy / Tara Pande / Marie La Russa / Lei S. Qi

    Cell Reports Medicine, Vol 2, Iss 4, Pp 100245- (2021)

    2021  

    Abstract: Summary: The coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) and variants has led to significant mortality. We recently reported that an RNA-targeting CRISPR-Cas13 system, called ... ...

    Abstract Summary: The coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) and variants has led to significant mortality. We recently reported that an RNA-targeting CRISPR-Cas13 system, called prophylactic antiviral CRISPR in human cells (PAC-MAN), offered an antiviral strategy against SARS-CoV-2 and influenza A virus. Here, we expand in silico analysis to use PAC-MAN to target a broad spectrum of human- or livestock-infectious RNA viruses with high specificity, coverage, and predicted efficiency. Our analysis reveals that a minimal set of 14 CRISPR RNAs (crRNAs) is able to target >90% of human-infectious viruses across 10 RNA virus families. We predict that a set of 5 experimentally validated crRNAs can target new SARS-CoV-2 variant sequences with zero mismatches. We also build an online resource (crispr-pacman.stanford.edu) to support community use of CRISPR-Cas13 for broad-spectrum RNA virus targeting. Our work provides a new bioinformatic resource for using CRISPR-Cas13 to target diverse RNA viruses to facilitate the development of CRISPR-based antivirals.
    Keywords CRISPR ; Cas13 ; SARS-CoV-2 ; COVID-19 ; antiviral ; RNA virus ; Medicine (General) ; R5-920
    Language English
    Publishing date 2021-04-01T00:00:00Z
    Publisher Elsevier
    Document type Article ; Online
    Database BASE - Bielefeld Academic Search Engine (life sciences selection)

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  4. Article ; Online: A benchmark of algorithms for the analysis of pooled CRISPR screens

    Sunil Bodapati / Timothy P. Daley / Xueqiu Lin / James Zou / Lei S. Qi

    Genome Biology, Vol 21, Iss 1, Pp 1-

    2020  Volume 13

    Abstract: Abstract Genome-wide pooled CRISPR-Cas-mediated knockout, activation, and repression screens are powerful tools for functional genomic investigations. Despite their increasing importance, there is currently little guidance on how to design and analyze ... ...

    Abstract Abstract Genome-wide pooled CRISPR-Cas-mediated knockout, activation, and repression screens are powerful tools for functional genomic investigations. Despite their increasing importance, there is currently little guidance on how to design and analyze CRISPR-pooled screens. Here, we provide a review of the commonly used algorithms in the computational analysis of pooled CRISPR screens. We develop a comprehensive simulation framework to benchmark and compare the performance of these algorithms using both synthetic and real datasets. Our findings inform parameter choices of CRISPR screens and provide guidance to researchers on the design and analysis of pooled CRISPR screens.
    Keywords CRISPR screen ; CRISPR knockoout ; CRISPR interference ; CRISPR activation ; Benchmarking ; Screen algorithms ; Biology (General) ; QH301-705.5 ; Genetics ; QH426-470
    Language English
    Publishing date 2020-03-01T00:00:00Z
    Publisher BMC
    Document type Article ; Online
    Database BASE - Bielefeld Academic Search Engine (life sciences selection)

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  5. Article ; Online: Identification of cell context-dependent YAP-associated proteins reveals β1 and β4 integrin mediate YAP translocation independently of cell spreading

    Joanna Y. Lee / Antonia A. Dominguez / Sungmin Nam / Ryan S. Stowers / Lei. S Qi / Ovijit Chaudhuri

    Scientific Reports, Vol 9, Iss 1, Pp 1-

    2019  Volume 11

    Abstract: Abstract Yes-associated protein (YAP) is a transcriptional regulator and mechanotransducer, relaying extracellular matrix (ECM) stiffness into proliferative gene expression in 2D culture. Previous studies show that YAP activation is dependent on F-actin ... ...

    Abstract Abstract Yes-associated protein (YAP) is a transcriptional regulator and mechanotransducer, relaying extracellular matrix (ECM) stiffness into proliferative gene expression in 2D culture. Previous studies show that YAP activation is dependent on F-actin stress fiber mediated nuclear pore opening, however the protein mediators of YAP translocation remain unclear. Here, we show that YAP co-localizes with F-actin during activating conditions, such as sparse plating and culturing on stiff 2D substrates. To identify proteins mediating YAP translocation, we performed co-immunoprecipitation followed by mass spectrometry (co-IP/MS) for proteins that differentially associated with YAP under activating conditions. Interestingly, YAP preferentially associates with β1 integrin under activating conditions, and β4 integrin under inactivating conditions. In activating conditions, CRISPR/Cas9 knockout (KO) of β1 integrin (ΔITGB1) resulted in decreased cell area, which correlated with decreased YAP nuclear localization. ΔITGB1 did not significantly affect the slope of the correlation between YAP nuclear localization with area, but did decrease overall nuclear YAP independently of cell spreading. In contrast, β4 integrin KO (ΔITGB4) cells showed no change in cell area and similarly decreased nuclear YAP. These results reveal proteins that differentially associate with YAP during activation, which may aid in regulating YAP nuclear translocation.
    Keywords Medicine ; R ; Science ; Q
    Subject code 570
    Language English
    Publishing date 2019-11-01T00:00:00Z
    Publisher Nature Publishing Group
    Document type Article ; Online
    Database BASE - Bielefeld Academic Search Engine (life sciences selection)

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  6. Article ; Online: CRISPhieRmix

    Timothy P. Daley / Zhixiang Lin / Xueqiu Lin / Yanxia Liu / Wing Hung Wong / Lei S. Qi

    Genome Biology, Vol 19, Iss 1, Pp 1-

    a hierarchical mixture model for CRISPR pooled screens

    2018  Volume 13

    Abstract: Abstract Pooled CRISPR screens allow researchers to interrogate genetic causes of complex phenotypes at the genome-wide scale and promise higher specificity and sensitivity compared to competing technologies. Unfortunately, two problems exist, ... ...

    Abstract Abstract Pooled CRISPR screens allow researchers to interrogate genetic causes of complex phenotypes at the genome-wide scale and promise higher specificity and sensitivity compared to competing technologies. Unfortunately, two problems exist, particularly for CRISPRi/a screens: variability in guide efficiency and large rare off-target effects. We present a method, CRISPhieRmix, that resolves these issues by using a hierarchical mixture model with a broad-tailed null distribution. We show that CRISPhieRmix allows for more accurate and powerful inferences in large-scale pooled CRISPRi/a screens. We discuss key issues in the analysis and design of screens, particularly the number of guides needed for faithful full discovery.
    Keywords CRISPR screen ; CRISPR interference ; CRISPR activation ; sgRNA design ; Mixture models ; Local fdr ; Biology (General) ; QH301-705.5 ; Genetics ; QH426-470
    Language English
    Publishing date 2018-10-01T00:00:00Z
    Publisher BMC
    Document type Article ; Online
    Database BASE - Bielefeld Academic Search Engine (life sciences selection)

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  7. Article ; Online: Transient non-integrative expression of nuclear reprogramming factors promotes multifaceted amelioration of aging in human cells

    Tapash Jay Sarkar / Marco Quarta / Shravani Mukherjee / Alex Colville / Patrick Paine / Linda Doan / Christopher M. Tran / Constance R. Chu / Steve Horvath / Lei S. Qi / Nidhi Bhutani / Thomas A. Rando / Vittorio Sebastiano

    Nature Communications, Vol 11, Iss 1, Pp 1-

    2020  Volume 12

    Abstract: Aging involves gradual loss of tissue function, and transcription factor (TF) expression can ameliorate this in progeroid mice. Here the authors show that transient TF expression reverses age-associated epigenetic marks, inflammatory profiles and ... ...

    Abstract Aging involves gradual loss of tissue function, and transcription factor (TF) expression can ameliorate this in progeroid mice. Here the authors show that transient TF expression reverses age-associated epigenetic marks, inflammatory profiles and restores regenerative potential in naturally aged human cells.
    Keywords Science ; Q
    Language English
    Publishing date 2020-03-01T00:00:00Z
    Publisher Nature Publishing Group
    Document type Article ; Online
    Database BASE - Bielefeld Academic Search Engine (life sciences selection)

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  8. Article ; Online: Transient non-integrative expression of nuclear reprogramming factors promotes multifaceted amelioration of aging in human cells

    Tapash Jay Sarkar / Marco Quarta / Shravani Mukherjee / Alex Colville / Patrick Paine / Linda Doan / Christopher M. Tran / Constance R. Chu / Steve Horvath / Lei S. Qi / Nidhi Bhutani / Thomas A. Rando / Vittorio Sebastiano

    Nature Communications, Vol 11, Iss 1, Pp 1-

    2020  Volume 12

    Abstract: Aging involves gradual loss of tissue function, and transcription factor (TF) expression can ameliorate this in progeroid mice. Here the authors show that transient TF expression reverses age-associated epigenetic marks, inflammatory profiles and ... ...

    Abstract Aging involves gradual loss of tissue function, and transcription factor (TF) expression can ameliorate this in progeroid mice. Here the authors show that transient TF expression reverses age-associated epigenetic marks, inflammatory profiles and restores regenerative potential in naturally aged human cells.
    Keywords Science ; Q
    Language English
    Publishing date 2020-03-01T00:00:00Z
    Publisher Nature Portfolio
    Document type Article ; Online
    Database BASE - Bielefeld Academic Search Engine (life sciences selection)

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  9. Article ; Online: YAP-independent mechanotransduction drives breast cancer progression

    Joanna Y. Lee / Jessica K. Chang / Antonia A. Dominguez / Hong-pyo Lee / Sungmin Nam / Julie Chang / Sushama Varma / Lei S. Qi / Robert B. West / Ovijit Chaudhuri

    Nature Communications, Vol 10, Iss 1, Pp 1-

    2019  Volume 9

    Abstract: The transcriptional regulator YAP is regarded as the universal mechanotransducer, largely from 2D culture studies. Here the authors show that in breast cancer patient tissues and cells in 3D culture, mechanical signals are transduced independently of YAP, ...

    Abstract The transcriptional regulator YAP is regarded as the universal mechanotransducer, largely from 2D culture studies. Here the authors show that in breast cancer patient tissues and cells in 3D culture, mechanical signals are transduced independently of YAP, questioning YAP as a therapeutic target.
    Keywords Science ; Q
    Language English
    Publishing date 2019-04-01T00:00:00Z
    Publisher Nature Publishing Group
    Document type Article ; Online
    Database BASE - Bielefeld Academic Search Engine (life sciences selection)

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  10. Article ; Online: YAP-independent mechanotransduction drives breast cancer progression

    Joanna Y. Lee / Jessica K. Chang / Antonia A. Dominguez / Hong-pyo Lee / Sungmin Nam / Julie Chang / Sushama Varma / Lei S. Qi / Robert B. West / Ovijit Chaudhuri

    Nature Communications, Vol 10, Iss 1, Pp 1-

    2019  Volume 9

    Abstract: The transcriptional regulator YAP is regarded as the universal mechanotransducer, largely from 2D culture studies. Here the authors show that in breast cancer patient tissues and cells in 3D culture, mechanical signals are transduced independently of YAP, ...

    Abstract The transcriptional regulator YAP is regarded as the universal mechanotransducer, largely from 2D culture studies. Here the authors show that in breast cancer patient tissues and cells in 3D culture, mechanical signals are transduced independently of YAP, questioning YAP as a therapeutic target.
    Keywords Science ; Q
    Language English
    Publishing date 2019-04-01T00:00:00Z
    Publisher Nature Portfolio
    Document type Article ; Online
    Database BASE - Bielefeld Academic Search Engine (life sciences selection)

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