While the catalog of mammalian transcripts and their expression levels in different cell types and disease states is rapidly expanding, our understanding of transcript function lags behind. We ...present a robust technology enabling systematic investigation of the cellular consequences of repressing or inducing individual transcripts. We identify rules for specific targeting of transcriptional repressors (CRISPRi), typically achieving 90%–99% knockdown with minimal off-target effects, and activators (CRISPRa) to endogenous genes via endonuclease-deficient Cas9. Together they enable modulation of gene expression over a ∼1,000-fold range. Using these rules, we construct genome-scale CRISPRi and CRISPRa libraries, each of which we validate with two pooled screens. Growth-based screens identify essential genes, tumor suppressors, and regulators of differentiation. Screens for sensitivity to a cholera-diphtheria toxin provide broad insights into the mechanisms of pathogen entry, retrotranslocation and toxicity. Our results establish CRISPRi and CRISPRa as powerful tools that provide rich and complementary information for mapping complex pathways.
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•CRISPRi and CRISPRa provide complementary information for mapping complex pathways•CRISPRi/a expression series (up to ∼1,000-fold) reveal how gene dose controls function•CRISPRi provides strong (typically 90%–99%) knockdown with minimal off-target effects•Genome-scale screens elucidate pathways controlling cholera/diphtheria toxicity
Genome-scale-specific targeting of transcriptional repressors (CRISPRi) and activators (CRISPRa) to endogenous genes via endonuclease-deficient Cas9 have been applied to growth and toxin-resistance screens, establishing CRISPRi and CRISPRa as powerful tools that provide rich and complementary information.
The genetic interrogation and reprogramming of cells requires methods for robust and precise targeting of genes for expression or repression. The CRISPR-associated catalytically inactive dCas9 ...protein offers a general platform for RNA-guided DNA targeting. Here, we show that fusion of dCas9 to effector domains with distinct regulatory functions enables stable and efficient transcriptional repression or activation in human and yeast cells, with the site of delivery determined solely by a coexpressed short guide (sg)RNA. Coupling of dCas9 to a transcriptional repressor domain can robustly silence expression of multiple endogenous genes. RNA-seq analysis indicates that CRISPR interference (CRISPRi)-mediated transcriptional repression is highly specific. Our results establish that the CRISPR system can be used as a modular and flexible DNA-binding platform for the recruitment of proteins to a target DNA sequence, revealing the potential of CRISPRi as a general tool for the precise regulation of gene expression in eukaryotic cells.
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•CRISPRi enables robust gene repression and activation in human cells•CRISPRi knockdown is specific with minimal off-target effects in human cells•CRISPRi can effectively repress endogenous genes in human and yeast•dCas9 enables modular and programmable RNA-guided genome regulation in eukaryotes
Catalytically inactive CRISPR can be targeted to specific loci in human and yeast cells to specifically repress and activate transcription. The study demonstrates the potential for adapting CRISPRi for multiple modes of transcriptional control, chromatin modification, and regulatory element mapping in a broad range of eukaryotes.
The spatiotemporal organization and dynamics of chromatin play critical roles in regulating genome function. However, visualizing specific, endogenous genomic loci remains challenging in living ...cells. Here, we demonstrate such an imaging technique by repurposing the bacterial CRISPR/Cas system. Using an EGFP-tagged endonuclease-deficient Cas9 protein and a structurally optimized small guide (sg) RNA, we show robust imaging of repetitive elements in telomeres and coding genes in living cells. Furthermore, an array of sgRNAs tiling along the target locus enables the visualization of nonrepetitive genomic sequences. Using this method, we have studied telomere dynamics during elongation or disruption, the subnuclear localization of the MUC4 loci, the cohesion of replicated MUC4 loci on sister chromatids, and their dynamic behaviors during mitosis. This CRISPR imaging tool has potential to significantly improve the capacity to study the conformation and dynamics of native chromosomes in living human cells.
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•An optimized CRISPR enables live imaging and better gene regulation in human cells•CRISPR imaging visualizes either repetitive or nonrepetitive genomic sequences•CRISPR imaging reports telomere length change and telomere movements•CRISPR imaging monitors the dynamics of gene loci throughout the cell cycle
A new CRISPR-based technology allows precise visualization of individual gene loci in living cells.
Targeted gene regulation on a genome-wide scale is a powerful strategy for interrogating, perturbing, and engineering cellular systems. Here, we develop a method for controlling gene expression based ...on Cas9, an RNA-guided DNA endonuclease from a type II CRISPR system. We show that a catalytically dead Cas9 lacking endonuclease activity, when coexpressed with a guide RNA, generates a DNA recognition complex that can specifically interfere with transcriptional elongation, RNA polymerase binding, or transcription factor binding. This system, which we call CRISPR interference (CRISPRi), can efficiently repress expression of targeted genes in Escherichia coli, with no detectable off-target effects. CRISPRi can be used to repress multiple target genes simultaneously, and its effects are reversible. We also show evidence that the system can be adapted for gene repression in mammalian cells. This RNA-guided DNA recognition platform provides a simple approach for selectively perturbing gene expression on a genome-wide scale.
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► Inactive CRISPR associated 9 protein (dCas9) is repurposed for genome engineering ► dCas9 and a complementary short guide RNA can target specific genomic sites ► CRISPR interference (CRISPRi) can regulate multiple genes without off-target effects ► CRISPRi is compact and can be ported to bacterial and mammalian cells
The authors have developed a CRISPR interference system in which a catalytically dead Cas9 protein can be targeted to a specific genomic site through a complementary small guide RNA, allowing systematic perturbation of gene transcription in bacteria and mammalian cells.
Signals in many biological processes can be amplified by recruiting multiple copies of regulatory proteins to a site of action. Harnessing this principle, we have developed a protein scaffold, a ...repeating peptide array termed SunTag, which can recruit multiple copies of an antibody-fusion protein. We show that the SunTag can recruit up to 24 copies of GFP, thereby enabling long-term imaging of single protein molecules in living cells. We also use the SunTag to create a potent synthetic transcription factor by recruiting multiple copies of a transcriptional activation domain to a nuclease-deficient CRISPR/Cas9 protein and demonstrate strong activation of endogenous gene expression and re-engineered cell behavior with this system. Thus, the SunTag provides a versatile platform for multimerizing proteins on a target protein scaffold and is likely to have many applications in imaging and controlling biological outputs.
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•SunTag allows controlled protein multimerization on a protein scaffold•SunTag enables long-term single-molecule imaging in living cells•SunTag greatly improves CRISPR-based activation of gene expression
A protein tagging system, SunTag, is developed to enable long-term single-molecule imaging and to enhance CRISPR-based transcriptional activation in living cells.
Cancer therapy targets malignant cells that are surrounded by a diverse complement of nonmalignant stromal cells. Therapy-induced damage of normal cells can alter the tumor microenvironment, causing ...cellular senescence and activating cancer-promoting inflammation. However, how these damage responses are regulated (both induced and resolved) to preserve tissue homeostasis and prevent chronic inflammation is poorly understood. Here, we detail an acute chemotherapy-induced secretory response that is self-limiting in vitro and in vivo despite the induction of cellular senescence. We used tissue-specific knockout mice to demonstrate that endothelial production of the proinflammatory cytokine IL-6 promotes chemoresistance and show that the chemotherapeutic doxorubicin induces acute IL-6 release through reactive oxygen species-mediated p38 activation in vitro. Doxorubicin causes endothelial senescence but, surprisingly, without a typical senescence secretory response. We found that endothelial cells repress senescence-associated inflammation through the down-regulation of PI3K/AKT/mTOR signaling and that reactivation of this pathway restores senescence-associated inflammation. Thus, we describe a mechanism by which damage-associated paracrine secretory responses are restrained to preserve tissue homeostasis and prevent chronic inflammation.
Developing technologies for efficient and scalable disruption of gene expression will provide powerful tools for studying gene function, developmental pathways, and disease mechanisms. Here, we ...develop clustered regularly interspaced short palindromic repeat interference (CRISPRi) to repress gene expression in human induced pluripotent stem cells (iPSCs). CRISPRi, in which a doxycycline-inducible deactivated Cas9 is fused to a KRAB repression domain, can specifically and reversibly inhibit gene expression in iPSCs and iPSC-derived cardiac progenitors, cardiomyocytes, and T lymphocytes. This gene repression system is tunable and has the potential to silence single alleles. Compared with CRISPR nuclease (CRISPRn), CRISPRi gene repression is more efficient and homogenous across cell populations. The CRISPRi system in iPSCs provides a powerful platform to perform genome-scale screens in a wide range of iPSC-derived cell types, dissect developmental pathways, and model disease.
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•Inducible CRISPRi iPSCs provide a valuable resource for rapid gene knockdown•CRISPRi knockdown is efficient, tunable, and reversible in iPSCs•CRISPRi knockdown is highly specific•CRISPRi enables disease modeling in iPSC-derived cardiomyocytes
In this article, Mandegar and colleagues utilize CRISPR interference for efficient gene knockdown in iPSCs and their differentiated cell derivatives. The CRISPRi tools and cell lines presented in this study are highly versatile and serve as a useful resource for the cell and stem cell biology communities.
In addition to the popular method of fluorescent protein fusion, live cell protein imaging has now seen more and more application of epitope tags. The small size of these tags may reduce functional ...perturbation and enable signal amplification. To address their background issue, we adapt self-complementing split fluorescent proteins as epitope tags for live cell protein labelling. The two tags, GFP11 and sfCherry11 are derived from the eleventh β-strand of super-folder GFP and sfCherry, respectively. The small size of FP11-tags enables a cost-effective and scalable way to insert them into endogenous genomic loci via CRISPR-mediated homology-directed repair. Tandem arrangement FP11-tags allows proportional enhancement of fluorescence signal in tracking intraflagellar transport particles, or reduction of photobleaching for live microtubule imaging. Finally, we show the utility of tandem GFP11-tag in scaffolding protein oligomerization. These experiments illustrate the versatility of FP11-tag as a labelling tool as well as a multimerization-control tool for both imaging and non-imaging applications.
How cellular and organismal complexity emerges from combinatorial expression of genes is a central question in biology. High-content phenotyping approaches such as Perturb-seq (single-cell ...RNA-sequencing pooled CRISPR screens) present an opportunity for exploring such genetic interactions (GIs) at scale. Here, we present an analytical framework for interpreting high-dimensional landscapes of cell states (manifolds) constructed from transcriptional phenotypes. We applied this approach to Perturb-seq profiling of strong GIs mined from a growth-based, gain-of-function GI map. Exploration of this manifold enabled ordering of regulatory pathways, principled classification of GIs (e.g., identifying suppressors), and mechanistic elucidation of synergistic interactions, including an unexpected synergy between
and
driving erythroid differentiation. Finally, we applied recommender system machine learning to predict interactions, facilitating exploration of vastly larger GI manifolds.
Eukaryotic cells execute complex transcriptional programs in which specific loci throughout the genome are regulated in distinct ways by targeted regulatory assemblies. We have applied this principle ...to generate synthetic CRISPR-based transcriptional programs in yeast and human cells. By extending guide RNAs to include effector protein recruitment sites, we construct modular scaffold RNAs that encode both target locus and regulatory action. Sets of scaffold RNAs can be used to generate synthetic multigene transcriptional programs in which some genes are activated and others are repressed. We apply this approach to flexibly redirect flux through a complex branched metabolic pathway in yeast. Moreover, these programs can be executed by inducing expression of the dCas9 protein, which acts as a single master regulatory control point. CRISPR-associated RNA scaffolds provide a powerful way to construct synthetic gene expression programs for a wide range of applications, including rewiring cell fates or engineering metabolic pathways.
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•CRISPR scaffold RNAs (scRNAs) encode both target locus and regulatory function•scRNAs function efficiently in mammalian and yeast cells•scRNAs enable transcription programs with simultaneous activation and repression•Combinatorial control of multiple genes enables flexible pathway manipulation
Modular CRISPR RNA scaffolds engineered to encode both guides to a target locus and recruitment of transcriptional regulators allow simultaneous gene activation and repression of multiple different genes in eukaryotic cells, greatly expanding the synthetic biology toolkit.
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