Project description:To fully exploit the microbial genome resources, a high-throughput experimental platform is needed to associate genes with phenotypes at the genome level. We present here a novel method that enables investigation of the cellular consequences of repressing individual transcripts based on the CRISPR interference (CRISPRi) pooled screening in bacteria. We identify rules for guide RNA library design to handle the unique structure of prokaryotic genomes by tiling screening and construct an E. coli genome-scale guide RNA library (~60,000 members) accordingly. We show that CRISPRi outperforms transposon sequencing, the benchmark method in the microbial functional genomics field, when similar library sizes are used or gene length is short. This tool is also effective for mapping phenotypes to non-coding RNAs (ncRNAs), as elucidated by a comprehensive tRNA-fitness map constructed here. Our results establish CRISPRi pooled screening as a powerful tool for mapping complex prokaryotic genetic networks in a precise and high-throughput manner.
Project description:Large-scale genetic interaction (GI) screens in yeast have been invaluable for our understanding of molecular systems biology and for characterizing novel gene function. Owing in part to the high costs and long experiment times required, a preponderance of GI data has been generated in a single environmental condition. However, an unknown fraction of GIs may be specific to other conditions. Here, we developed a pooled-growth CRISPRi-based sequencing assay for GIs, CRISPRiSeq, which increases throughput such that GIs can be easily assayed across multiple growth conditions. We assayed the fitness of approximately 17,000 strains encompassing approximately 7700 pairwise interactions in five conditions and found that the additional conditions increased the number of GIs detected nearly threefold over the number detected in rich media alone. In addition, we found that condition-specific GIs are prevalent and improved the power to functionally classify genes. Finally, we found new links during respiratory growth between members of the Ras nutrient-sensing pathway and both the COG complex and a gene of unknown function. Our results highlight the potential of conditional GI screens to improve our understanding of cellular genetic networks.
Project description: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.
Project description:Targeted gene regulation is indispensable for reprogramming a cellular network to modulate a microbial phenotype. Here, we adopted the type II CRISPR interference (CRISPRi) system for simple and efficient regulation of target genes in Pseudomonas putida KT2440. A single CRISPRi plasmid was generated to express a nuclease-deficient Cas9 gene and a designed single guide RNA, under control of l-rhamnose-inducible Prha BAD and the constitutive Biobrick J23119 promoter respectively. Two target genes were selected to probe the CRISPRi-mediated gene regulation: exogenous green fluorescent protein on the multicopy plasmid and endogenous glpR on the P. putida KT2440 chromosome, encoding GlpR, a transcriptional regulator that represses expression of the glpFKRD gene cluster for glycerol utilization. The CRISPRi system successfully repressed the two target genes, as evidenced by a reduction in the fluorescence intensity and the lag phase of P. putida KT2440 cell growth on glycerol. Furthermore, CRISPRi-mediated repression of glpR improved both the cell growth and glycerol utilization, resulting in the enhanced production of mevalonate in an engineered P. putida KT2440 harbouring heterologous genes for the mevalonate pathway. CRISPRi is expected to become a robust tool to reprogram P. putida KT2440 for the development of microbial cell factories producing industrially valuable products.
Project description:Pooled CRISPR-Cas9 screens are a powerful method for functionally characterizing regulatory elements in the non-coding genome, but off-target effects in these experiments have not been systematically evaluated. Here, we investigate Cas9, dCas9, and CRISPRi/a off-target activity in screens for essential regulatory elements. The sgRNAs with the largest effects in genome-scale screens for essential CTCF loop anchors in K562 cells were not single guide RNAs (sgRNAs) that disrupted gene expression near the on-target CTCF anchor. Rather, these sgRNAs had high off-target activity that, while only weakly correlated with absolute off-target site number, could be predicted by the recently developed GuideScan specificity score. Screens conducted in parallel with CRISPRi/a, which do not induce double-stranded DNA breaks, revealed that a distinct set of off-targets also cause strong confounding fitness effects with these epigenome-editing tools. Promisingly, filtering of CRISPRi libraries using GuideScan specificity scores removed these confounded sgRNAs and enabled identification of essential regulatory elements.
Project description: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.
Project description:A recent genome-wide screen identified ~300 essential or growth-supporting genes in the dental caries pathogen Streptococcus mutans. To be able to study these genes, we built a CRISPR interference tool around the Cas9 nuclease (Cas9Smu) encoded in the S. mutans UA159 genome. Using a xylose-inducible dead Cas9Smu with a constitutively active single-guide RNA (sgRNA), we observed titratable repression of GFP fluorescence that compared favorably to that of Streptococcus pyogenes dCas9 (Cas9Spy). We then investigated sgRNA specificity and proto-spacer adjacent motif (PAM) requirements. Interference by sgRNAs did not occur with double or triple base-pair mutations, or if single base-pair mutations were in the 3' end of the sgRNA. Bioinformatic analysis of >450 S. mutans genomes allied with in vivo assays revealed a similar PAM recognition sequence as Cas9Spy. Next, we created a comprehensive library of sgRNA plasmids that were directed at essential and growth-supporting genes. We discovered growth defects for 77% of the CRISPRi strains expressing sgRNAs. Phenotypes of CRISPRi strains, across several biological pathways, were assessed using fluorescence microscopy. A variety of cell structure anomalies were observed, including segregational instability of the chromosome, enlarged cells, and ovococci-to-rod shape transitions. CRISPRi was also employed to observe how silencing of cell wall glycopolysaccharide biosynthesis (rhamnose-glucose polysaccharide, RGP) affected both cell division and pathogenesis in a wax worm model. The CRISPRi tool and sgRNA library are valuable resources for characterizing essential genes in S. mutans, some of which could prove to be promising therapeutic targets.
Project description:CRISPR interference (CRISPRi) using dCas9-sgRNA is a powerful tool for the exploration and manipulation of gene functions. Here we quantify the reversible switching of a central process of the bacterial cell cycle by CRISPRi and an antisense RNA mechanism. Reversible induction of filamentous growth in E. coli has been recently demonstrated by controlling the expression levels of the bacterial cell division proteins FtsZ/FtsA via CRISPRi. If FtsZ falls below a critical level, cells cannot divide. However, the cells remain metabolically active and continue with DNA replication. We surmised that this makes them amenable to an inducible antisense RNA strategy to counteract FtsZ inhibition. We show that both static and inducible thresholds can adjust the characteristics of the switching process. Combining bulk data with single cell measurements, we characterize the efficiency of the switching process. Successful restoration of division is found to occur faster in the presence of antisense sgRNAs than upon simple termination of CRISPRi induction.
Project description:Genome-wide screens have discovered a large set of essential genes in the opportunistic human pathogen Streptococcus pneumoniae However, the functions of many essential genes are still unknown, hampering vaccine development and drug discovery. Based on results from transposon sequencing (Tn-seq), we refined the list of essential genes in S. pneumoniae serotype 2 strain D39. Next, we created a knockdown library targeting 348 potentially essential genes by CRISPR interference (CRISPRi) and show a growth phenotype for 254 of them (73%). Using high-content microscopy screening, we searched for essential genes of unknown function with clear phenotypes in cell morphology upon CRISPRi-based depletion. We show that SPD_1416 and SPD_1417 (renamed to MurT and GatD, respectively) are essential for peptidoglycan synthesis, and that SPD_1198 and SPD_1197 (renamed to TarP and TarQ, respectively) are responsible for the polymerization of teichoic acid (TA) precursors. This knowledge enabled us to reconstruct the unique pneumococcal TA biosynthetic pathway. CRISPRi was also employed to unravel the role of the essential Clp-proteolytic system in regulation of competence development, and we show that ClpX is the essential ATPase responsible for ClpP-dependent repression of competence. The CRISPRi library provides a valuable tool for characterization of pneumococcal genes and pathways and revealed several promising antibiotic targets.
Project description:The rapid adoption of CRISPR technology has enabled biomedical researchers to conduct CRISPR-based genetic screens in a pooled format. The quality of results from such screens is heavily dependent on the selection of optimal screen design parameters, which also affects cost and scalability. However, the cost and effort of implementing pooled screens prohibits experimental testing of a large number of parameters.We present CRISPulator, a Monte Carlo method-based computational tool that simulates the impact of screen parameters on the robustness of screen results, thereby enabling users to build intuition and insights that will inform their experimental strategy. CRISPulator enables the simulation of screens relying on either CRISPR interference (CRISPRi) or CRISPR nuclease (CRISPRn). Pooled screens based on cell growth/survival, as well as fluorescence-activated cell sorting according to fluorescent reporter phenotypes are supported. CRISPulator is freely available online ( http://crispulator.ucsf.edu ).CRISPulator facilitates the design of pooled genetic screens by enabling the exploration of a large space of experimental parameters in silico, rather than through costly experimental trial and error. We illustrate its power by deriving non-obvious rules for optimal screen design.