Publications by authors named "Lei S Qi"

55 Publications

Engineering 3D genome organization.

Nat Rev Genet 2021 Feb 8. Epub 2021 Feb 8.

Department of Bioengineering, Stanford University, Stanford, CA, USA.

Cancers and developmental disorders are associated with alterations in the 3D genome architecture in space and time (the fourth dimension). Mammalian 3D genome organization is complex and dynamic and plays an essential role in regulating gene expression and cellular function. To study the causal relationship between genome function and its spatio-temporal organization in the nucleus, new technologies for engineering and manipulating the 3D organization of the genome have been developed. In particular, CRISPR-Cas technologies allow programmable manipulation at specific genomic loci, enabling unparalleled opportunities in this emerging field of 3D genome engineering. We review advances in mammalian 3D genome engineering with a focus on recent manipulative technologies using CRISPR-Cas and related technologies.
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http://dx.doi.org/10.1038/s41576-020-00325-5DOI Listing
February 2021

CRISPR technologies for precise epigenome editing.

Nat Cell Biol 2021 01 8;23(1):11-22. Epub 2021 Jan 8.

Department of Bioengineering, Stanford University, Stanford, CA, USA.

The epigenome involves a complex set of cellular processes governing genomic activity. Dissecting this complexity necessitates the development of tools capable of specifically manipulating these processes. The repurposing of prokaryotic CRISPR systems has allowed for the development of diverse technologies for epigenome engineering. Here, we review the state of currently achievable epigenetic manipulations along with corresponding applications. With future optimization, CRISPR-based epigenomic editing stands as a set of powerful tools for understanding and controlling biological function.
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http://dx.doi.org/10.1038/s41556-020-00620-7DOI Listing
January 2021

Regenerating Urethral Striated Muscle by CRISPRi/dCas9-KRAB-Mediated Myostatin Silencing for Obesity-Associated Stress Urinary Incontinence.

CRISPR J 2020 Dec;3(6):562-572

Knuppe Molecular Urology Laboratory, Department of Urology, School of Medicine, University of California, San Francisco, California, USA; Department of Chemical and Systems Biology, ChEM-H, Stanford University, Stanford, California, USA.

Overweight females are prone to obesity-associated stress urinary incontinence (OA-SUI), and there are no definitive medical therapies for this common urologic condition. This study was designed to test the hypothesis that regenerative therapy to restore urethral striated muscle (stM) and pelvic floor muscles might represent a valuable therapeutic approach. For the experiment, single-guide RNAs targeting myostatin () were used for CRISPRi/dCas9-Kruppel associated box (KRAB)-mediated gene silencing. For the experiment, a total of 14 female lean ZUC-Lepr 186 and 14 fatty ZUC-Lepr 185 rats were used as control and CRISPRi-MSTN treated groups, respectively. The results indicated that lentivirus-mediated expression of MSTN CRISPRi/dCas9-KRAB caused sustained downregulation of MSTN in rat L6 myoblast cells and significantly enhanced myogenesis . , the urethral sphincter injection of lentiviral-MSTN sgRNA and lentiviral-dCas9-KRAB significantly increased the leak point pressure, the thickness of the stM layer, the ratio of stM to smooth muscle, and the number of neuromuscular junctions. Downregulation of with CRISPRi/dCas9-KRAB-mediated gene silencing significantly enhanced myogenesis and It also improved urethral continence in the OA-SUI rat model.
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http://dx.doi.org/10.1089/crispr.2020.0077DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7757699PMC
December 2020

Double Emulsion Picoreactors for High-Throughput Single-Cell Encapsulation and Phenotyping via FACS.

Anal Chem 2020 10 23;92(19):13262-13270. Epub 2020 Sep 23.

Department of Bioengineering, Stanford University, Stanford, California 94305, United States.

In the past five years, droplet microfluidic techniques have unlocked new opportunities for the high-throughput genome-wide analysis of single cells, transforming our understanding of cellular diversity and function. However, the field lacks an accessible method to screen and sort droplets based on cellular phenotype upstream of genetic analysis, particularly for large and complex cells. To meet this need, we developed Dropception, a robust, easy-to-use workflow for precise single-cell encapsulation into picoliter-scale double emulsion droplets compatible with high-throughput screening via fluorescence-activated cell sorting (FACS). We demonstrate the capabilities of this method by encapsulating five standardized mammalian cell lines of varying sizes and morphologies as well as a heterogeneous cell mixture of a whole dissociated flatworm (5-25 μm in diameter) within highly monodisperse double emulsions (35 μm in diameter). We optimize for preferential encapsulation of single cells with extremely low multiple-cell loading events (<2% of cell-containing droplets), thereby allowing direct linkage of cellular phenotype to genotype. Across all cell lines, cell loading efficiency approaches the theoretical limit with no observable bias by cell size. FACS measurements reveal the ability to discriminate empty droplets from those containing cells with good agreement to single-cell occupancies quantified via microscopy, establishing robust droplet screening at single-cell resolution. High-throughput FACS screening of cellular picoreactors has the potential to shift the landscape of single-cell droplet microfluidics by expanding the repertoire of current nucleic acid droplet assays to include functional phenotyping.
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http://dx.doi.org/10.1021/acs.analchem.0c02499DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7670281PMC
October 2020

Low-frequency ultrasound-mediated cytokine transfection enhances T cell recruitment at local and distant tumor sites.

Proc Natl Acad Sci U S A 2020 06 19;117(23):12674-12685. Epub 2020 May 19.

Molecular Imaging Program at Stanford, Stanford University, Stanford, CA 94305;

Robust cytotoxic T cell infiltration has proven to be difficult to achieve in solid tumors. We set out to develop a flexible protocol to efficiently transfect tumor and stromal cells to produce immune-activating cytokines, and thus enhance T cell infiltration while debulking tumor mass. By combining ultrasound with tumor-targeted microbubbles, membrane pores are created and facilitate a controllable and local transfection. Here, we applied a substantially lower transmission frequency (250 kHz) than applied previously. The resulting microbubble oscillation was significantly enhanced, reaching an effective expansion ratio of 35 for a peak negative pressure of 500 kPa in vitro. Combining low-frequency ultrasound with tumor-targeted microbubbles and a DNA plasmid construct, 20% of tumor cells remained viable, and ∼20% of these remaining cells were transfected with a reporter gene both in vitro and in vivo. The majority of cells transfected in vivo were mucin 1/CD45 tumor cells. Tumor and stromal cells were then transfected with plasmid DNA encoding IFN-β, producing 150 pg/10 cells in vitro, a 150-fold increase compared to no-ultrasound or no-plasmid controls and a 50-fold increase compared to treatment with targeted microbubbles and ultrasound (without IFN-β). This enhancement in secretion exceeds previously reported fourfold to fivefold increases with other in vitro treatments. Combined with intraperitoneal administration of checkpoint inhibition, a single application of IFN-β plasmid transfection reduced tumor growth in vivo and recruited efficacious immune cells at both the local and distant tumor sites.
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http://dx.doi.org/10.1073/pnas.1914906117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7293655PMC
June 2020

Development of CRISPR as an Antiviral Strategy to Combat SARS-CoV-2 and Influenza.

Cell 2020 05 29;181(4):865-876.e12. Epub 2020 Apr 29.

Department of Bioengineering, Stanford University, Stanford, CA 94305, USA; Department of Chemical and Systems Biology, Stanford University, Stanford, CA 94305, USA; ChEM-H, Stanford University, Stanford, CA 94305, USA. Electronic address:

The coronavirus disease 2019 (COVID-19) pandemic, caused by the SARS-CoV-2 virus, has highlighted the need for antiviral approaches that can target emerging viruses with no effective vaccines or pharmaceuticals. Here, we demonstrate a CRISPR-Cas13-based strategy, PAC-MAN (prophylactic antiviral CRISPR in human cells), for viral inhibition that can effectively degrade RNA from SARS-CoV-2 sequences and live influenza A virus (IAV) in human lung epithelial cells. We designed and screened CRISPR RNAs (crRNAs) targeting conserved viral regions and identified functional crRNAs targeting SARS-CoV-2. This approach effectively reduced H1N1 IAV load in respiratory epithelial cells. Our bioinformatic analysis showed that a group of only six crRNAs can target more than 90% of all coronaviruses. With the development of a safe and effective system for respiratory tract delivery, PAC-MAN has the potential to become an important pan-coronavirus inhibition strategy.
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http://dx.doi.org/10.1016/j.cell.2020.04.020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7189862PMC
May 2020

Transient non-integrative expression of nuclear reprogramming factors promotes multifaceted amelioration of aging in human cells.

Nat Commun 2020 03 24;11(1):1545. Epub 2020 Mar 24.

Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, Stanford, CA, 94305, USA.

Aging is characterized by a gradual loss of function occurring at the molecular, cellular, tissue and organismal levels. At the chromatin level, aging associates with progressive accumulation of epigenetic errors that eventually lead to aberrant gene regulation, stem cell exhaustion, senescence, and deregulated cell/tissue homeostasis. Nuclear reprogramming to pluripotency can revert both the age and the identity of any cell to that of an embryonic cell. Recent evidence shows that transient reprogramming can ameliorate age-associated hallmarks and extend lifespan in progeroid mice. However, it is unknown how this form of rejuvenation would apply to naturally aged human cells. Here we show that transient expression of nuclear reprogramming factors, mediated by expression of mRNAs, promotes a rapid and broad amelioration of cellular aging, including resetting of epigenetic clock, reduction of the inflammatory profile in chondrocytes, and restoration of youthful regenerative response to aged, human muscle stem cells, in each case without abolishing cellular identity.
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http://dx.doi.org/10.1038/s41467-020-15174-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7093390PMC
March 2020

Fibrinogen Alpha Chain Knockout Promotes Tumor Growth and Metastasis through Integrin-AKT Signaling Pathway in Lung Cancer.

Mol Cancer Res 2020 07 23;18(7):943-954. Epub 2020 Mar 23.

Department of Genetics, University of Alabama at Birmingham, Birmingham, Alabama.

Fibrinogen is an extracellular matrix protein composed of three polypeptide chains with fibrinogen alpha (FGA), beta (FGB) and gamma (FGG). Although fibrinogen and its related fragments are involved in tumor angiogenesis and metastasis, their functional roles are incompatible. A recent genome-scale screening reveals that loss of affects the acceleration of tumor growth and metastasis of lung cancer, but the mechanism remains elusive. We used CRISPR/Cas9 genome editing to knockout (KO) in human lung adenocarcinoma (LUAD) cell lines A549 and H1299. By colony formation, transwell migration and matrix invasion assays, KO increased cell proliferation, migration, and invasion but decreased the expressions of epithelial-mesenchymal transition marker E-cadherin and cytokeratin 5/8 in A549 and H1299 cells. However, administration of FGA inhibited cell proliferation and migration but induced apoptosis in A549 cells. Of note, KO cells indirectly cocultured by transwells with wild-type cells increased FGA in the culture medium, leading to decreased migration of KO cells. Furthermore, our functional analysis identified a direct interaction of FGA with integrin α5 as well as FGA-integrin signaling that regulated the AKT-mTOR signaling pathway in A549 cells. In addition, we validated that KO increased tumor growth and metastasis through activation of AKT signaling in an A549 xenograft model. IMPLICATIONS: These findings demonstrate that that loss of facilities tumor growth and metastasis through the integrin-AKT signaling pathway in lung cancer.
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http://dx.doi.org/10.1158/1541-7786.MCR-19-1033DOI Listing
July 2020

A benchmark of algorithms for the analysis of pooled CRISPR screens.

Genome Biol 2020 03 9;21(1):62. Epub 2020 Mar 9.

Department of Bioengineering, Stanford University, 450 Serra Mall, Stanford, 94305, USA.

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.
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http://dx.doi.org/10.1186/s13059-020-01972-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7063732PMC
March 2020

Therapeutic genome editing in cardiovascular diseases.

Adv Drug Deliv Rev 2021 01 21;168:147-157. Epub 2020 Feb 21.

Stanford Cardiovascular Institute, Stanford University School of Medicine, Stanford, CA 94305, United States; Division of Cardiovascular Medicine, Department of Medicine, Stanford University School of Medicine, Stanford, CA 94305, United States; Department of Radiology, Stanford University School of Medicine, Stanford, CA 94305, United States. Electronic address:

During the past decade, developments in genome editing technology have fundamentally transformed biomedical research. In particular, the CRISPR/Cas9 system has been extensively applied because of its simplicity and ability to alter genomic sequences within living organisms, and an ever increasing number of CRISPR/Cas9-based molecular tools are being developed for a wide variety of applications. While genome editing tools have been used for many aspects of biological research, they also have enormous potential to be used for genome editing therapy to treat a broad range of diseases. For some hematopoietic diseases, clinical trials of therapeutic genome editing with CRISPR/Cas9 are already starting phase I. In the cardiovascular field, genome editing tools have been utilized to understand the mechanisms of diseases such as cardiomyopathy, arrythmia, and lipid metabolism, which now open the door to therapeutic genome editing. Currently, therapeutic genome editing in the cardiovascular field is centered on liver-targeting strategies to reduce cardiovascular risks. Targeting the heart is more challenging. In this review, we discuss the potential applications, recent advances, and current limitations of therapeutic genome editing in the cardiovascular field.
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http://dx.doi.org/10.1016/j.addr.2020.02.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7442585PMC
January 2021

Multiple Input Sensing and Signal Integration Using a Split Cas12a System.

Mol Cell 2020 04 5;78(1):184-191.e3. Epub 2020 Feb 5.

Department of Bioengineering, Stanford University, Stanford, CA 94305, USA; Department of Chemical and Systems Biology, Stanford University, Stanford, CA 94305, USA; ChEM-H, Stanford University, Stanford, CA 94305, USA. Electronic address:

The ability to integrate biological signals and execute a functional response when appropriate is critical for sophisticated cell engineering using synthetic biology. Although the CRISPR-Cas system has been harnessed for synthetic manipulation of the genome, it has not been fully utilized for complex environmental signal sensing, integration, and actuation. Here, we develop a split dCas12a platform and show that it allows for the construction of multi-input, multi-output logic circuits in mammalian cells. The system is highly programmable and can generate expandable AND gates with two, three, and four inputs. It can also incorporate NOT logic by using anti-CRISPR proteins as an OFF switch. By coupling the split dCas12a design to multiple tumor-relevant promoters, we provide a proof of concept that the system can implement logic gating to specifically detect breast cancer cells and execute therapeutic immunomodulatory responses.
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http://dx.doi.org/10.1016/j.molcel.2020.01.016DOI Listing
April 2020

Identification of cell context-dependent YAP-associated proteins reveals β and β integrin mediate YAP translocation independently of cell spreading.

Sci Rep 2019 11 20;9(1):17188. Epub 2019 Nov 20.

Department of Mechanical Engineering, Stanford University, Stanford, CA, 94305, USA.

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 β integrin under activating conditions, and β integrin under inactivating conditions. In activating conditions, CRISPR/Cas9 knockout (KO) of β 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, β 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.
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http://dx.doi.org/10.1038/s41598-019-53659-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6868278PMC
November 2019

CRISPR-mediated live imaging of genome editing and transcription.

Science 2019 09 5;365(6459):1301-1305. Epub 2019 Sep 5.

Department of Bioengineering, Stanford University, Stanford, CA 94305, USA.

We report a robust, versatile approach called CRISPR live-cell fluorescent in situ hybridization (LiveFISH) using fluorescent oligonucleotides for genome tracking in a broad range of cell types, including primary cells. An intrinsic stability switch of CRISPR guide RNAs enables LiveFISH to accurately detect chromosomal disorders such as Patau syndrome in prenatal amniotic fluid cells and track multiple loci in human T lymphocytes. In addition, LiveFISH tracks the real-time movement of DNA double-strand breaks induced by CRISPR-Cas9-mediated editing and consequent chromosome translocations. Finally, by combining Cas9 and Cas13 systems, LiveFISH allows for simultaneous visualization of genomic DNA and RNA transcripts in living cells. The LiveFISH approach enables real-time live imaging of DNA and RNA during genome editing, transcription, and rearrangements in single cells.
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http://dx.doi.org/10.1126/science.aax7852DOI Listing
September 2019

Site-Programmable Transposition: Shifting the Paradigm for CRISPR-Cas Systems.

Mol Cell 2019 07;75(2):206-208

Department of Bioengineering, Stanford University, Stanford, CA, USA; Department of Chemical and Systems Biology, Stanford University, Stanford, CA, USA; ChEM-H, Stanford University, Stanford, CA, USA. Electronic address:

Discoveries by Klompe et al. (2019) and Strecker et al. (2019) elucidate distinct CRISPR-Cas mechanisms for site-specific programmable transposition in prokaryotic organisms.
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http://dx.doi.org/10.1016/j.molcel.2019.07.004DOI Listing
July 2019

Reversible Disruption of Specific Transcription Factor-DNA Interactions Using CRISPR/Cas9.

Mol Cell 2019 05;74(3):622-633.e4

Department of Biology, Stanford University, Stanford, CA 94305, USA. Electronic address:

The control of gene expression by transcription factor binding sites frequently determines phenotype. However, it is difficult to determine the function of single transcription factor binding sites within larger transcription networks. Here, we use deactivated Cas9 (dCas9) to disrupt binding to specific sites, a method we term CRISPRd. Since CRISPR guide RNAs are longer than transcription factor binding sites, flanking sequence can be used to target specific sites. Targeting dCas9 to an Oct4 site in the Nanog promoter displaced Oct4 from this site, reduced Nanog expression, and slowed division. In contrast, disrupting the Oct4 binding site adjacent to Pax6 upregulated Pax6 transcription and disrupting Nanog binding its own promoter upregulated its transcription. Thus, we can easily distinguish between activating and repressing binding sites and examine autoregulation. Finally, multiple guide RNA expression allows simultaneous inhibition of multiple binding sites, and conditionally destabilized dCas9 allows rapid reversibility.
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http://dx.doi.org/10.1016/j.molcel.2019.04.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6599634PMC
May 2019

YAP-independent mechanotransduction drives breast cancer progression.

Nat Commun 2019 04 23;10(1):1848. Epub 2019 Apr 23.

Department of Mechanical Engineering, Stanford University, Stanford, CA, 94305, USA.

Increased tissue stiffness is a driver of breast cancer progression. The transcriptional regulator YAP is considered a universal mechanotransducer, based largely on 2D culture studies. However, the role of YAP during in vivo breast cancer remains unclear. Here, we find that mechanotransduction occurs independently of YAP in breast cancer patient samples and mechanically tunable 3D cultures. Mechanistically, the lack of YAP activity in 3D culture and in vivo is associated with the absence of stress fibers and an order of magnitude decrease in nuclear cross-sectional area relative to 2D culture. This work highlights the context-dependent role of YAP in mechanotransduction, and establishes that YAP does not mediate mechanotransduction in breast cancer.
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http://dx.doi.org/10.1038/s41467-019-09755-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6478686PMC
April 2019

When genome editing goes off-target.

Science 2019 04 18;364(6437):234-236. Epub 2019 Apr 18.

Department of Bioengineering, Stanford University, Stanford, CA 94305, USA.

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http://dx.doi.org/10.1126/science.aax1827DOI Listing
April 2019

Identification of Novel Regulatory Genes in APAP Induced Hepatocyte Toxicity by a Genome-Wide CRISPR-Cas9 Screen.

Sci Rep 2019 02 4;9(1):1396. Epub 2019 Feb 4.

Division of Experimental and Translational Genetics, University of Missouri Kansas City School of Medicine, Kansas City, USA.

Acetaminophen (APAP) is a commonly used analgesic responsible for more than half of acute liver failure cases. Identification of previously unknown genetic risk factors would provide mechanistic insights and novel therapeutic targets for APAP-induced liver injury. This study used a genome-wide CRISPR-Cas9 screen to evaluate genes that are protective against, or cause susceptibility to, APAP-induced liver injury. HuH7 human hepatocellular carcinoma cells containing CRISPR-Cas9 gene knockouts were treated with 15 mM APAP for 30 minutes to 4 days. A gene expression profile was developed based on the 1) top screening hits, 2) overlap of expression data from APAP overdose studies, and 3) predicted affected biological pathways. We further demonstrated the implementation of intermediate time points for the identification of early and late response genes. This study illustrated the power of a genome-wide CRISPR-Cas9 screen to systematically identify novel genes involved in APAP-induced hepatotoxicity and to provide potential targets to develop novel therapeutic modalities.
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http://dx.doi.org/10.1038/s41598-018-37940-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6362041PMC
February 2019

Anti-CRISPR-mediated control of gene editing and synthetic circuits in eukaryotic cells.

Nat Commun 2019 01 14;10(1):194. Epub 2019 Jan 14.

Department of Bioengineering, Stanford University, Stanford, CA, 94305, USA.

Repurposed CRISPR-Cas molecules provide a useful tool set for broad applications of genomic editing and regulation of gene expression in prokaryotes and eukaryotes. Recent discovery of phage-derived proteins, anti-CRISPRs, which serve to abrogate natural CRISPR anti-phage activity, potentially expands the ability to build synthetic CRISPR-mediated circuits. Here, we characterize a panel of anti-CRISPR molecules for expanded applications to counteract CRISPR-mediated gene activation and repression of reporter and endogenous genes in various cell types. We demonstrate that cells pre-engineered with anti-CRISPR molecules become resistant to gene editing, thus providing a means to generate "write-protected" cells that prevent future gene editing. We further show that anti-CRISPRs can be used to control CRISPR-based gene regulation circuits, including implementation of a pulse generator circuit in mammalian cells. Our work suggests that anti-CRISPR proteins should serve as widely applicable tools for synthetic systems regulating the behavior of eukaryotic cells.
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http://dx.doi.org/10.1038/s41467-018-08158-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6331597PMC
January 2019

Evolution at the Cutting Edge: CRISPR-Mediated Directed Evolution.

Mol Cell 2018 11;72(3):402-403

Department of Bioengineering, Stanford University, Stanford, CA 94305, USA; Department of Chemical and Systems Biology, Stanford University, Stanford, CA 94305, USA; Stanford ChEM-H, Stanford University, Stanford, CA 94305, USA. Electronic address:

In a recent issue of Nature, Halperin et al. (2018) develop a new technology to continuously diversify specific genomic loci by combining CRISPR-Cas9 with error-prone DNA polymerases.
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http://dx.doi.org/10.1016/j.molcel.2018.10.027DOI Listing
November 2018

CRISPR Activation Screens Systematically Identify Factors that Drive Neuronal Fate and Reprogramming.

Cell Stem Cell 2018 11 11;23(5):758-771.e8. Epub 2018 Oct 11.

Department of Bioengineering, Stanford University, Stanford, CA 94305, USA; Department of Chemical and Systems Biology, Stanford University, Stanford, CA 94305, USA; Stanford ChEM-H, Stanford University, Stanford, CA 94305, USA. Electronic address:

Comprehensive identification of factors that can specify neuronal fate could provide valuable insights into lineage specification and reprogramming, but systematic interrogation of transcription factors, and their interactions with each other, has proven technically challenging. We developed a CRISPR activation (CRISPRa) approach to systematically identify regulators of neuronal-fate specification. We activated expression of all endogenous transcription factors and other regulators via a pooled CRISPRa screen in embryonic stem cells, revealing genes including epigenetic regulators such as Ezh2 that can induce neuronal fate. Systematic CRISPR-based activation of factor pairs allowed us to generate a genetic interaction map for neuronal differentiation, with confirmation of top individual and combinatorial hits as bona fide inducers of neuronal fate. Several factor pairs could directly reprogram fibroblasts into neurons, which shared similar transcriptional programs with endogenous neurons. This study provides an unbiased discovery approach for systematic identification of genes that drive cell-fate acquisition.
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http://dx.doi.org/10.1016/j.stem.2018.09.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6214761PMC
November 2018

CRISPR-Mediated Programmable 3D Genome Positioning and Nuclear Organization.

Cell 2018 11 11;175(5):1405-1417.e14. Epub 2018 Oct 11.

Department of Bioengineering, Stanford University, Stanford, CA 94305, USA; Department of Chemical and Systems Biology, Stanford University, Stanford, CA 94305, USA; Stanford ChEM-H, Stanford University, Stanford, CA 94305, USA. Electronic address:

Programmable control of spatial genome organization is a powerful approach for studying how nuclear structure affects gene regulation and cellular function. Here, we develop a versatile CRISPR-genome organization (CRISPR-GO) system that can efficiently control the spatial positioning of genomic loci relative to specific nuclear compartments, including the nuclear periphery, Cajal bodies, and promyelocytic leukemia (PML) bodies. CRISPR-GO is chemically inducible and reversible, enabling interrogation of real-time dynamics of chromatin interactions with nuclear compartments in living cells. Inducible repositioning of genomic loci to the nuclear periphery allows for dissection of mitosis-dependent and -independent relocalization events and also for interrogation of the relationship between gene position and gene expression. CRISPR-GO mediates rapid de novo formation of Cajal bodies at desired chromatin loci and causes significant repression of endogenous gene expression over long distances (30-600 kb). The CRISPR-GO system offers a programmable platform to investigate large-scale spatial genome organization and function.
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http://dx.doi.org/10.1016/j.cell.2018.09.013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6239909PMC
November 2018

CRISPhieRmix: a hierarchical mixture model for CRISPR pooled screens.

Genome Biol 2018 10 8;19(1):159. Epub 2018 Oct 8.

Department of Bioengineering, Stanford University, 443 Via Ortega, Stanford, 94305, USA.

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.
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http://dx.doi.org/10.1186/s13059-018-1538-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6176515PMC
October 2018

A CRISPR-dCas Toolbox for Genetic Engineering and Synthetic Biology.

Authors:
Xiaoshu Xu Lei S Qi

J Mol Biol 2019 01 26;431(1):34-47. Epub 2018 Jun 26.

Department of Bioengineering, Stanford University, Stanford, CA 94305, USA; Department of Chemical and Systems Biology, Stanford University, Stanford, CA 94305, USA; Stanford ChEM-H Institute, Stanford University, Stanford, CA 94305, USA. Electronic address:

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.
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http://dx.doi.org/10.1016/j.jmb.2018.06.037DOI Listing
January 2019

CRISPR-Based Chromatin Remodeling of the Endogenous Oct4 or Sox2 Locus Enables Reprogramming to Pluripotency.

Cell Stem Cell 2018 02 18;22(2):252-261.e4. Epub 2018 Jan 18.

The J. David Gladstone Institutes, 1650 Owens Street, San Francisco, CA 94158, USA; School of Pharmaceutical Sciences, Tsinghua University, Beijing 100084, China. Electronic address:

Generation of induced pluripotent stem cells typically requires the ectopic expression of transcription factors to reactivate the pluripotency network. However, it remains largely unclear what remodeling events on endogenous chromatin trigger reprogramming toward induced pluripotent stem cells (iPSCs). Toward this end, we employed CRISPR activation to precisely target and remodel endogenous gene loci of Oct4 and Sox2. Interestingly, we found that single-locus targeting of Sox2 was sufficient to remodel and activate Sox2, which was followed by the induction of other pluripotent genes and establishment of the pluripotency network. Simultaneous remodeling of the Oct4 promoter and enhancer also triggered reprogramming. Authentic pluripotent cell lines were established in both cases. Finally, we showed that targeted manipulation of histone acetylation at the Oct4 gene locus could also initiate reprogramming. Our study generated authentic iPSCs with CRISPR activation through precise epigenetic remodeling of endogenous loci and shed light on how targeted chromatin remodeling triggers pluripotency induction.
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http://dx.doi.org/10.1016/j.stem.2017.12.001DOI Listing
February 2018

Engineering cell sensing and responses using a GPCR-coupled CRISPR-Cas system.

Nat Commun 2017 12 20;8(1):2212. Epub 2017 Dec 20.

Department of Bioengineering, Stanford University, Stanford, CA, 94305, USA.

G-protein-coupled receptors (GPCRs) are the largest and most diverse group of membrane receptors in eukaryotes and detect a wide array of cues in the human body. Here we describe a molecular device that couples CRISPR-dCas9 genome regulation to diverse natural and synthetic extracellular signals via GPCRs. We generate alternative architectures for fusing CRISPR to GPCRs utilizing the previously reported design, Tango, and our design, ChaCha. Mathematical modeling suggests that for the CRISPR ChaCha design, multiple dCas9 molecules can be released across the lifetime of a GPCR. The CRISPR ChaCha is dose-dependent, reversible, and can activate multiple endogenous genes simultaneously in response to extracellular ligands. We adopt the design to diverse GPCRs that sense a broad spectrum of ligands, including synthetic compounds, chemokines, mitogens, fatty acids, and hormones. This toolkit of CRISPR-coupled GPCRs provides a modular platform for rewiring diverse ligand sensing to targeted genome regulation for engineering cellular functions.
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http://dx.doi.org/10.1038/s41467-017-02075-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5738360PMC
December 2017

A Single-Chain Photoswitchable CRISPR-Cas9 Architecture for Light-Inducible Gene Editing and Transcription.

ACS Chem Biol 2018 02 29;13(2):443-448. Epub 2017 Sep 29.

Department of Bioengineering, Stanford University , Stanford, California, United States.

Optical control of CRISPR-Cas9-derived proteins would be useful for restricting gene editing or transcriptional regulation to desired times and places. Optical control of Cas9 functions has been achieved with photouncageable unnatural amino acids or by using light-induced protein interactions to reconstitute Cas9-mediated functions from two polypeptides. However, these methods have only been applied to one Cas9 species and have not been used for optical control of different perturbations at two genes. Here, we use photodissociable dimeric fluorescent protein domains to engineer single-chain photoswitchable Cas9 (ps-Cas9) proteins in which the DNA-binding cleft is occluded at baseline and opened upon illumination. This design successfully controlled different species and functional variants of Cas9, mediated transcriptional activation more robustly than previous optogenetic methods, and enabled light-induced transcription of one gene and editing of another in the same cells. Thus, a single-chain photoswitchable architecture provides a general method to control a variety of Cas9-mediated functions.
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http://dx.doi.org/10.1021/acschembio.7b00603DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5820652PMC
February 2018

Genetic interaction mapping in mammalian cells using CRISPR interference.

Nat Methods 2017 Jun 8;14(6):577-580. Epub 2017 May 8.

Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, California, USA.

We describe a combinatorial CRISPR interference (CRISPRi) screening platform for mapping genetic interactions in mammalian cells. We targeted 107 chromatin-regulation factors in human cells with pools of either single or double single guide RNAs (sgRNAs) to downregulate individual genes or gene pairs, respectively. Relative enrichment analysis of individual sgRNAs or sgRNA pairs allowed for quantitative characterization of genetic interactions, and comparison with protein-protein-interaction data revealed a functional map of chromatin regulation.
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http://dx.doi.org/10.1038/nmeth.4286DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5584685PMC
June 2017

Multiplexed Dynamic Imaging of Genomic Loci by Combined CRISPR Imaging and DNA Sequential FISH.

Biophys J 2017 May 17;112(9):1773-1776. Epub 2017 Apr 17.

Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, California. Electronic address:

Visualization of chromosome dynamics allows the investigation of spatiotemporal chromatin organization and its role in gene regulation and other cellular processes. However, current approaches to label multiple genomic loci in live cells have a fundamental limitation in the number of loci that can be labeled and uniquely identified. Here we describe an approach we call "track first and identify later" for multiplexed visualization of chromosome dynamics by combining two techniques: CRISPR imaging and DNA sequential fluorescence in situ hybridization. Our approach first labels and tracks chromosomal loci in live cells with the CRISPR-Cas9 system, then barcodes those loci by DNA sequential fluorescence in situ hybridization in fixed cells and resolves their identities. We demonstrate our approach by tracking telomere dynamics, identifying 12 unique subtelomeric regions with variable detection efficiencies, and tracking back the telomere dynamics of respective chromosomes in mouse embryonic stem cells.
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http://dx.doi.org/10.1016/j.bpj.2017.03.024DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5425380PMC
May 2017