Luke A. Gilbert

CRISPR-Mediated Modular RNA-Guided Regulation of Transcription in Eukaryotes

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.

A Protein-Tagging System for Signal Amplification in Gene Expression and Fluorescence Imaging

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.

Engineering Complex Synthetic Transcriptional Programs with CRISPR RNA Scaffolds

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.

CRISPR interference (CRISPRi) for sequence-specific control of gene expression

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 CRISPR (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 sgRNAs 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.

Toxoplasma gondii Targets a Protein Phosphatase 2C to the Nuclei of Infected Host Cells

ABSTRACT Intracellular pathogens have evolved a wide array of mechanisms to invade and co-opt their host cells for intracellular survival. Apicomplexan parasites such as Toxoplasma gondii employ the action of unique secretory organelles named rhoptries for internalization of the parasite and formation of a specialized niche within the host cell. We demonstrate that Toxoplasma gondii also uses secretion from the rhoptries during invasion to deliver a parasite-derived protein phosphatase 2C (PP2C-hn) into the host cell and direct it to the host nucleus. Delivery to the host nucleus does not require completion of invasion, as evidenced by the fact that parasites blocked in the initial stages of invasion with cytochalasin D are able to target PP2C-hn to the host nucleus. We have disrupted the gene encoding PP2C-hn and shown that PP2C-hn-knockout parasites exhibit a mild growth defect that can be rescued by complementation with the wild-type gene. The delivery of parasite effector proteins via the rhoptries provides a novel mechanism for Toxoplasma to directly access the command center of its host cell during infection by the parasite.

CRISPRi-based genome-scale identification of functional long noncoding RNA loci in human cells

A very focused function for lncRNAs The human genome generates many thousands of long noncoding RNAs (lncRNAs). A very small number of lncRNAs have been shown to be functional. Liu et al. carried out a large-scale CRISPR-based screen to assess the function of ∼17,000 lncRNAs in seven different human cell lines. A considerable number (∼500) of the tested lncRNAs influenced cell growth, suggesting biological function. In almost all cases, though, the function was highly cell type—specific, often limited to just one cell type. Science, this issue p. 10.1126/science.aah7111 A considerable fraction of long noncoding RNAs have highly cell type–specific biological functions. INTRODUCTION The human genome contains tens of thousands of loci that produce long noncoding RNAs (lncRNAs), transcripts that have no apparent protein-coding potential. A subset of lncRNAs have been found to play critical roles in cellular processes, organismal development, and disease. Although these examples are suggestive of the importance and diversity of lncRNAs, the vast majority of lncRNA genes have not been functionally tested. RATIONALE Because it is currently not possible to predict which lncRNA loci are functional or what function they perform, there is a need for large-scale, systematic approaches to interrogating the functional contribution of lncRNA loci. We therefore developed a genome-scale screening platform based on CRISPR-mediated interference (CRISPRi), which uses a catalytically inactive CRISPR effector protein, (d)Cas9, fused to a repressive KRAB domain and targeted by a single guide RNA (sgRNA), to inhibit gene expression. By catalyzing repressive chromatin modifications around the transcription start site (TSS) and serving as a transcriptional roadblock, CRISPRi tests a broad range of lncRNA gene functions, including the production of cis- and trans-acting RNA transcripts, cis-mediated regulation related to lncRNA transcription itself, and enhancer-like function of some lncRNA loci. RESULTS We designed a CRISPRi Non-Coding Library (CRiNCL), which targets 16,401 lncRNA genes each with 10 sgRNAs per TSS, and applied this pooled screening approach to identify lncRNA genes that modify robust cell growth. We screened seven human cell lines, including six transformed cell lines and induced pluripotent stem cells (iPSCs), and identified 499 lncRNA loci that modified cell growth upon CRISPRi targeting; 372 and 299 of these loci were distal from any protein coding gene or mapped enhancer, respectively. Extensive validation confirmed the screen results and demonstrated the robust and specific performance of CRISPRi for repressing lncRNA transcription. Remarkably, 89% of the lncRNA gene hits modified growth in just one of the cell lines tested, and no hits were common to all seven cell lines. Although nearly all of the hit genes were expressed in the cell line in which they exhibited a growth phenotype, expression alone was insufficient to explain the cell type specificity of their function. Transcriptional profiling revealed extensive gene expression changes upon CRISPRi targeting of lncRNA loci in the cells in which they modified growth, whereas targeting the same lncRNA locus in other cell lines resulted in minimal changes to the transcriptome beyond depletion of the targeted lncRNA transcript itself. CONCLUSION Our study considerably increases the number of known functional lncRNA loci. More broadly, our CRISPRi approach enables mechanistic studies of specific lncRNA functions and, when applied systematically, supports the global exploration of the complex biology contained in the lncRNA-expressing genome. Finally, in contrast to recent studies that found that essential protein-coding genes typically are required across a broad range of cell types, we show that lncRNA function is highly cell type–specific, a finding that has important implications for their involvement in both normal biology and disease. CRISPRi screening of lncRNAs in human cells. CRISPRi can precisely repress transcription of lncRNAs. The CRISPRi Non-Coding Library (CRiNCL) was generated to interrogate the function of thousands of long noncoding RNAs in seven different cell lines. Validation studies confirmed the exquisite cell type–specific function of lncRNAs. The human genome produces thousands of long noncoding RNAs (lncRNAs)—transcripts >200 nucleotides long that do not encode proteins. Although critical roles in normal biology and disease have been revealed for a subset of lncRNAs, the function of the vast majority remains untested. We developed a CRISPR interference (CRISPRi) platform targeting 16,401 lncRNA loci in seven diverse cell lines, including six transformed cell lines and human induced pluripotent stem cells (iPSCs). Large-scale screening identified 499 lncRNA loci required for robust cellular growth, of which 89% showed growth-modifying function exclusively in one cell type. We further found that lncRNA knockdown can perturb complex transcriptional networks in a cell type–specific manner. These data underscore the functional importance and cell type specificity of many lncRNAs.

Nucleosomes impede Cas9 access to DNA in vivo and in vitro

The prokaryotic CRISPR (clustered regularly interspaced palindromic repeats)-associated protein, Cas9, has been widely adopted as a tool for editing, imaging, and regulating eukaryotic genomes. However, our understanding of how to select single-guide RNAs (sgRNAs) that mediate efficient Cas9 activity is incomplete, as we lack insight into how chromatin impacts Cas9 targeting. To address this gap, we analyzed large-scale genetic screens performed in human cell lines using either nuclease-active or nuclease-dead Cas9 (dCas9). We observed that highly active sgRNAs for Cas9 and dCas9 were found almost exclusively in regions of low nucleosome occupancy. In vitro experiments demonstrated that nucleosomes in fact directly impede Cas9 binding and cleavage, while chromatin remodeling can restore Cas9 access. Our results reveal a critical role of eukaryotic chromatin in dictating the targeting specificity of this transplanted bacterial enzyme, and provide rules for selecting Cas9 target sites distinct from and complementary to those based on sequence properties. DOI: http://dx.doi.org/10.7554/eLife.12677.001

CRISPR Interference Efficiently Induces Specific and Reversible Gene Silencing in Human iPSCs

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.

Defining principles of combination drug mechanisms of action.

Combination chemotherapies have been a mainstay in the treatment of disseminated malignancies for almost 60 y, yet even successful regimens fail to cure many patients. Although their single-drug components are well studied, the mechanisms by which drugs work together in clinical combination regimens are poorly understood. Here, we combine RNAi-based functional signatures with complementary informatics tools to examine drug combinations. This approach seeks to bring to combination therapy what the knowledge of biochemical targets has brought to single-drug therapy and creates a statistical and experimental definition of “combination drug mechanisms of action.” We show that certain synergistic drug combinations may act as a more potent version of a single drug. Conversely, unlike these highly synergistic combinations, most drugs average extant single-drug variations in therapeutic response. When combined to form multidrug regimens, averaging combinations form averaging regimens that homogenize genetic variation in mouse models of cancer and in clinical genomics datasets. We suggest surprisingly simple and predictable combination mechanisms of action that are independent of biochemical mechanism and have implications for biomarker discovery as well as for the development of regimens with defined genetic dependencies.

Compact and highly active next-generation libraries for CRISPR-mediated gene repression and activation

We recently found that nucleosomes directly block access of CRISPR/Cas9 to DNA (Horlbeck et al., 2016). Here, we build on this observation with a comprehensive algorithm that incorporates chromatin, position, and sequence features to accurately predict highly effective single guide RNAs (sgRNAs) for targeting nuclease-dead Cas9-mediated transcriptional repression (CRISPRi) and activation (CRISPRa). We use this algorithm to design next-generation genome-scale CRISPRi and CRISPRa libraries targeting human and mouse genomes. A CRISPRi screen for essential genes in K562 cells demonstrates that the large majority of sgRNAs are highly active. We also find CRISPRi does not exhibit any detectable non-specific toxicity recently observed with CRISPR nuclease approaches. Precision-recall analysis shows that we detect over 90% of essential genes with minimal false positives using a compact 5 sgRNA/gene library. Our results establish CRISPRi and CRISPRa as premier tools for loss- or gain-of-function studies and provide a general strategy for identifying Cas9 target sites. DOI: http://dx.doi.org/10.7554/eLife.19760.001