SUMMARY 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.

SUMMARY 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 delivey determined solely by a co-expressed 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 and reveal the potential of CRISPRi as a general tool for the precise regulation of gene expression in eukaryotic cells.

SUMMARY Many signaling systems show adaptation—the ability to reset themselves after responding to a stimulus. We computationally searched all possible three-node enzyme network topologies to identify those that could perform adaptation. Only two major core topologies emerge as robust solutions: a negative feedback loop with a buffering node and an incoherent feedforward loop with a proportioner node. Minimal circuits containing these topologies are, within proper regions of parameter space, sufficient to achieve adaptation. Morecomplex circuits that robustly performadaptation all contain at least one of these topologies at their core. This analysis yields a design table highlighting a finite set of adaptive circuits. Despite the diversity of possible biochemical networks, it may be common to find that only a finite set of core topologies can execute a particular function. These design rules provide a framework for functionally classifying complex natural networks and a manual for engineering networks. For a video summary of this article, see the PaperFlick file with the Supplemental Data available online.

Choanoflagellates are the closest known relatives of metazoans. To discover potential molecular mechanisms underlying the evolution of metazoan multicellularity, we sequenced and analysed the genome of the unicellular choanoflagellate Monosiga brevicollis. The genome contains approximately 9,200 intron-rich genes, including a number that encode cell adhesion and signalling protein domains that are otherwise restricted to metazoans. Here we show that the physical linkages among protein domains often differ between M. brevicollis and metazoans, suggesting that abundant domain shuffling followed the separation of the choanoflagellate and metazoan lineages. The completion of the M. brevicollis genome allows us to reconstruct with increasing resolution the genomic changes that accompanied the origin of metazoans.Choanoflagellates have long fascinated evolutionary biologists for their marked similarity to the 'feeding cells' (choanocytes) of sponges and the possibility that they might represent the closest living relatives of metazoans 1,2 . Over the past decade or so, evidence supporting this relationship has accumulated from phylogenetic analyses of nuclear and mitochondrial genes [3][4][5][6] , comparative genomics between the mitochondrial genomes of choanoflagellates, sponges and other metazoans 7,8 , and the finding that choanoflagellates express homologues of metazoan signalling and adhesion genes 9-12 . Furthermore, species-rich phylogenetic analyses demonstrate that choanoflagellates are not derived from metazoans, but instead represent a distinct lineage that evolved before the origin and diversification of metazoans (Fig. 1a, Supplementary Fig. 1 and Supplementary Note 3.1) 8,13 . By virtue of their position on the tree of life, studies of choanoflagellates provide an unparallelled window into the nature of the unicellular and colonial progenitors of metazoans 14 .Choanoflagellates are abundant and globally distributed microbial eukaryotes found in marine and freshwater environments 15,16 . Like sponge choanocytes, each cell bears an apical flagellum surrounded by a distinctive collar of actin-filled microvilli, with which choanoflagellates trap bacteria and detritus (Fig. 1b). Using this highly effective means of prey capture, choanoflagellates link bacteria to higher trophic levels and thus have critical roles in oceanic carbon cycling and in the microbial food web 17,18 .More than 125 choanoflagellate species have been identified, and all species have a unicellular life-history stage. Some can also form simple colonies of equipotent cells, although these differ substantially from the obligate associations of differentiated cells in metazoans 19 . Studies of basal metazoans indicate that the ancestral metazoan was multicellular and had differentiated cell types, an epithelium, a body plan and regulated development including gastrulation. In contrast, the last common ancestor of choanoflagellates and metazoans was unicellular or possibly capable of forming simple colonies, underscoring the abundant biologi...

Genetically-encodable optical reporters, such as Green Fluorescent Protein, have revolutionized the observation and measurement of cellular states. However, the inverse challenge of using light to precisely control cellular behavior has only recently begun to be addressed; semi-synthetic chromophore-tethered receptors1 and naturally-occurring channel rhodopsins have been used to directly perturb neuronal networks2,3. The difficulty of engineering light sensitive proteins remains a significant impediment to the optical control to most cell-biological processes. Here we demonstrate the use of a new genetically-encoded light-control system based on an optimized reversible protein-protein interaction from the phytochrome signaling network of Arabidopsis thaliana. Because protein-protein interactions are one of the most general currencies of cellular information, this system can in principal be generically used to control diverse functions. Here we show that this system can be used to precisely and reversibly translocate target proteins to the membrane with micrometer spatial resolution and second time resolution. We show that light-gated translocation of the upstream activators of rho-family GTPases, which control the actin cytoskeleton, can be used to precisely reshape and direct the cell morphology of mammalian cells. The light-gated protein-protein interaction that has been optimized in this work should be useful for the design of diverse light-programmable reagents, potentially enabling a new generation of perturbative, quantitative experiments in cell biology.

Summary 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 multi-gene 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.

Sequence-specific control of gene expression on a genome-wide scale is an important approach for understanding gene functions and for engineering genetic regulatory systems. We have recently described an RNA-based method, CRISPR interference (CRISPRi), for targeted silencing of transcription in bacteria and human cells. The CRISPRi system is derived from the Streptococcus pyogenes CR ISPR (clustered regularly interspaced palindromic repeats) pathway, requiring only the coexpression of a catalytically inactive Cas9 protein and a customizable single guide RNA (sgRNA ). The Cas9-sgRNA complex binds to DNA elements complementary to the sgRNA and causes a steric block that halts transcript elongation by RNA polymerase, resulting in the repression of the target gene. Here we provide a protocol for the design, construction and expression of customized sgRNA s for transcriptional repression of any gene of interest. We also provide details for testing the repression activity of CRISPRi using quantitative fluorescence assays and native elongating transcript sequencing. CRISPRi provides a simplified approach for rapid gene repression within 1–2 weeks. The method can also be adapted for high-throughput interrogation of genome-wide gene functions and genetic interactions, thus providing a complementary approach to RNA interference, which can be used in a wider variety of organisms.

The spatial and temporal organization of molecules within a cell is critical for coordinating the many distinct activities carried out by the cell. In an increasing number of biological signaling processes, scaffold proteins have been found to play a central role in physically assembling the relevant molecular components. Although most scaffolds use a simple tethering mechanism to increase the efficiency of interaction between individual partner molecules, these proteins can also exert complex allosteric control over their partners and are themselves the target of regulation. Scaffold proteins offer a simple, flexible strategy for regulating selectivity in pathways, shaping output behaviors, and achieving new responses from preexisting signaling components. As a result, scaffold proteins have been exploited by evolution, pathogens, and cellular engineers to reshape cellular behavior.