Modeling predicts that CRISPR-based activators, unlike CRISPR-based repressors, scale well with increasing gRNA competition and dCas9 bottlenecking

Synthetic transcriptional networks built from CRISPR-based repressors (CRISPRi) rely on shared use of a core dCas9 protein. In E. coli, CRISPRi cannot sup- port more than about a dozen simultaneous gRNAs before the fold repression of any individual gRNA drops below 10x. We show with a simple model based on previous characterization of competition in CRISPRi that activation by CRISPR-based activators (CRISPRa) is much less sensitive to dCas9 bottle- necking than CRISPRi. We predict that E. coli should be able to support dozens to hundreds of CRISPRa gRNAs at > 10-fold activation

Ezh2-dCas9 and KRAB-dCas9 enable engineering of epigenetic memory in a context-dependent manner.

BackgroundRewriting of the epigenome has risen as a promising alternative to gene editing for precision medicine. In nature, epigenetic silencing can result in complete attenuation of target gene expression over multiple mitotic divisions. However, persistent repression has been difficult to achieve in a predictable manner using targeted systems.ResultsHere, we report that persistent epigenetic memory required both a DNA methyltransferase (DNMT3A-dCas9) and a histone methyltransferase (Ezh2-dCas9 or KRAB-dCas9). We demonstrate that the histone methyltransferase requirement can be locus specific. Co-targeting Ezh2-dCas9, but not KRAB-dCas9, with DNMT3A-dCas9 and DNMT3L induced long-term HER2 repression over at least 50 days (approximately 57 cell divisions) and triggered an epigenetic switch to a heterochromatic environment. An increase in H3K27 trimethylation and DNA methylation was stably maintained and accompanied by a sustained loss of H3K27 acetylation. Interestingly, substitution of Ezh2-dCas9 with KRAB-dCas9 enabled long-term repression at some target genes (e.g., SNURF) but not at HER2, at which H3K9me3 and DNA methylation were transiently acquired and subsequently lost. Off-target DNA hypermethylation occurred at many individual CpG sites but rarely at multiple CpGs in a single promoter, consistent with no detectable effect on transcription at the off-target loci tested. Conversely, robust hypermethylation was observed at HER2. We further demonstrated that Ezh2-dCas9 required full-length DNMT3L for maximal activity and that co-targeting DNMT3L was sufficient for persistent repression by Ezh2-dCas9 or KRAB-dCas9.ConclusionsThese data demonstrate that targeting different combinations of histone and DNA methyltransferases is required to achieve maximal repression at different loci. Fine-tuning of targeting tools is a necessity to engineer epigenetic memory at any given locus in any given cell type

Overcoming Metabolic Burden in Synthetic Biology: a CRISPR interference approach

Synthetic Biology is gaining an increasingly important role in the scientific community and dedicated research centers are rising all over the world. This discipline introduced the engineering principles of abstraction, modularity and standardization in the biology world; the application of these principles is allowing the design of complex biological systems to program living cells, realizing all sorts of desired function in many fields. These systems consist of DNA sequences, rationally combined to program the genetic instructions for cell behavior customization. Each part should behave as a biological brick for the design of complex genetic programs through functional building blocks; each module undergoes an extensive characterization to provide documentation on its functioning, enabling the rational design of complex circuits. Mathematical modeling accompanies all the design procedure as a tool to describe the behavior of each single genetic module, in a bottom-up fashion that should allow the prediction of more complex systems obtained by the interconnection of pre-characterized parts. However, many unpredictability sources hamper the ideally rational design of those synthetic genetic devices, mainly due to the tangled context-dependency behavior of those parts once placed into an intrinsically complex biological living system. Among others, the finite amount of translational resources in prokaryotic cells leads to an effect called metabolic burden, as a result of which hidden interactions between protein synthesis rates arise, leading to unexpected counterintuitive behaviors. To face this issue, two actions have been proposed in this study: firstly, a recently proposed mathematical modeling solution that included a description of the metabolic load exerted by the expression of recombinant genes have been applied on a case study, highlighting its worth of use and working boundaries; second, a CRISPR interference-based architecture have been developed to be used as an alternative to high resource usage transcriptional protein regulators, studying the underlying mechanism in several circuital configurations and optimizing each forming part in order to achieve the desired specifications. In Chapter 1, an introduction on synthetic biology is presented; in the second part, a brief overview on CRISPR technology and the overall aim of the study are reported. In Chapter 2, a case study evaluating the use of mathematical modeling to properly include metabolic burden in rational design of a set of transcriptional regulator cascades is reported. Firstly, the circuits and expected behavior are introduced, along with the discussion about experimental data, dissenting from what initially predicted. Secondly, the comparison between the use of a classical Hill equation-based model and an improved version that explicitly consider the translational load exerted by the expression of recombinant genes is reported. In Chapter 3, the design and deep characterization of a BioBrick$^{TM}$-compatible CRISPR interference-based repression set of modules is shown; expression optimization of the molecular players is reported and its usability as a low-burden alternative is demonstrated with experimental data and mathematical modeling. Working boundaries, peculiar aspects and rooms for improvements are then highlighted. In Chapter 4, preliminary studies aimed to improve the CRISPR interference system are reported and some of its context-dependencies are highlighted. Effects on repression efficiency due to alteration in the sequence of the RNA molecules addressing the CRISPR machinery to the desired target are discussed; evaluation of problems and opportunities related to the expression of more of this RNA guides are then highlighted. Lastly, an example of behavior of the system in presence of a competitor transcriptional regulator is reported. In Chapter 5 the overall conclusions of this thesis work are drawn

CRISPR-Cas9: a powerful tool to efficiently engineer Saccharomyces cerevisiae

Saccharomyces cerevisiae has been for a long time a common model for fundamental biological studies and a popular biotechnological engineering platform to produce chemicals, fuels, and pharmaceuticals due to its peculiar characteristics. Both lines of research require an effective editing of the native genetic elements or the inclusion of heterologous pathways into the yeast genome. Although S. cerevisiae is a well-known host with several molecular biology tools available, a more precise tool is still needed. The clustered, regularly interspaced, short palindromic repeats–associated Cas9 (CRISPR-Cas9) system is a current, widespread genome editing tool. The implementation of a reprogrammable, precise, and specific method, such as CRISPR-Cas9, to edit the S. cerevisiae genome has revolutionized laboratory practices. Herein, we describe and discuss some applications of the CRISPR-Cas9 system in S. cerevisiae from simple gene knockouts to more complex processes such as artificial heterologous pathway integration, transcriptional regulation, or tolerance engineering.This study was supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UIDB/BIO/04469/2020 unit and BioTecNorte operation (NORTE-01-0145-FEDER-000004) funded by the European Regional Development Fund (ERDF) under the scope of Norte2020—North Portugal Regional Program. In addition, this research has been carried out at the Biomass and Bioenergy Research Infrastructure (BBRI)—LISBOA-010145- FEDER-022059, supported by Operational Program for Competitiveness and Internationalization (PORTUGAL2020), the Lisbon Portugal Regional Operational Program (Lisboa2020), and Norte2020 under the Portugal 2020 Partnership Agreement, through the ERDF. J.R. is recipient of a fellowship supported by a doctoral advanced training (SFRH/BD/138325/2018) funded by FCT.info:eu-repo/semantics/publishedVersio

Modeling predicts that CRISPR-based activators, unlike CRISPR-based repressors, scale well with increasing gRNA competition and dCas9 bottlenecking

Synthetic transcriptional networks built from CRISPR-based repressors (CRISPRi) rely on shared use of a core dCas9 protein. In E. coli, CRISPRi cannot sup- port more than about a dozen simultaneous gRNAs before the fold repression of any individual gRNA drops below 10x. We show with a simple model based on previous characterization of competition in CRISPRi that activation by CRISPR-based activators (CRISPRa) is much less sensitive to dCas9 bottle- necking than CRISPRi. We predict that E. coli should be able to support dozens to hundreds of CRISPRa gRNAs at > 10-fold activation

Vulnerability and robustness in the essential gene complement of two bacterial species, profiled with CRISPRi

Bacterial essential genes contribute to the most fundamental processes of cellular life. The study of their functions in vivo has long been intractable to systematic genetic approaches, which are fundamental to understanding pathway level connections that govern cellular life and are a requirement for dissecting the complex cellular processes to which essential genes contribute. In Chapter 1 of this work I review recent advances in mapping gene-phenotype relationships in bacteria using the CRISPR-based technology, CRISPR interference (CRISPRi) for titratable gene knockdowns, focusing on their applications to the studies of essential genes, the exploration of chemical-genetic interactions, and the prospects for disentangling complex phenotypes in diverse bacterial species. In Chapter 2 I describe my analysis of the essential gene functions in the model Gram-negative bacterium Escherichia coli and the model Gram-positive Bacillus subtilis using datasets from paired chemical-genetic screens. In this work I identify both shared and Gram-negative specific mechanisms of collateral sensitization to antibiotic action. In Chapter 3 I investigate a fundamental property of essential genes, which is the relationship between their expression level and the cellular growth rate. Here, further developing CRISPRi tools in bacteria to predictably titrate knockdown efficacy, I interpret the knockdown-fitness relationships of each essential gene in E. coli and B. subtilis, discovering broad conservation of constraints setting and maintaining expression levels across these diverged species

A new age in functional genomics using CRISPR/Cas9 in arrayed library screening

CRISPR technology has rapidly changed the face of biological research, such that precise genome editing has now become routine for many labs within several years of its initial development. What makes CRISPR/Cas9 so revolutionary is the ability to target a protein (Cas9) to an exact genomic locus, through designing a specific short complementary nucleotide sequence, that together with a common scaffold sequence, constitute the guide RNA bridging the protein and the DNA. Wild-type Cas9 cleaves both DNA strands at its target sequence, but this protein can also be modified to exert many other functions. For instance, by attaching an activation domain to catalytically inactive Cas9 and targeting a promoter region, it is possible to stimulate the expression of a specific endogenous gene. In principle, any genomic region can be targeted, and recent efforts have successfully generated pooled guide RNA libraries for coding and regulatory regions of human, mouse and Drosophila genomes with high coverage, thus facilitating functional phenotypic screening. In this review, we will highlight recent developments in the area of CRISPR-based functional genomics and discuss potential future directions, with a special focus on mammalian cell systems and arrayed library screening

Development and application of CRISPR-based genetic tools in Bacillus species and Bacillus phages

Recently, the clustered regularly interspaced short palindromic repeats (CRISPR) system has been developed into a precise and efficient genome editing tool. Since its discovery as an adaptive immune system in prokaryotes, it has been applied in many different research fields including biotechnology and medical sciences. The high demand for rapid, highly efficient and versatile genetic tools to thrive in bacteria-based cell factories accelerates this process. This review mainly focuses on significant advancements of the CRISPR system in Bacillus subtilis, including the achievements in gene editing, and on problems still remaining. Next, we comprehensively summarize this genetic tool's up-to-date development and utilization in other Bacillus species, including B. licheniformis, B. methanolicus, B. anthracis, B. cereus, B. smithii and B. thuringiensis. Furthermore, we describe the current application of CRISPR tools in phages to increase Bacillus hosts' resistance to virulent phages and phage genetic modification. Finally, we suggest potential strategies to further improve this advanced technique and provide insights into future directions of CRISPR technologies for rendering Bacillus species cell factories more effective and more powerful

Biofuels from cyanobacteria -a metabolic engineering approach

The concern about the limited availability of petroleum-based fuels and their role in increasing CO2 levels in the atmosphere has sparked significant attention toward biofuel and bioenergy production. The global pursuit of sustainable energy sources has catalyzed innovative research into alternative biofuel production strategies. Transforming CO2 into usable fuels and chemicals is gaining even more prominence. Cyanobacteria, renowned for their photosynthetic ability, have emerged as promising candidates for biofuel synthesis. Their ability to convert solar energy and carbon dioxide into valuable biofuels makes them a compelling avenue for sustainable energy solutions. Using metabolic engineering principles, researchers have endeavored to optimize cyanobacterial metabolic pathways, enhance photosynthetic efficiency, and redirect carbon flux toward biofuel precursors. Numerous species of cyanobacteria offer genetic and metabolic traits that facilitate manipulation, and their photosynthetic characteristics imply that carbohydrates, fatty acids, and even alcohol could serve as potential renewable sources for biofuels. This review showcases cyanobacteria's ability as a biofuel source and emphasizes the transformative influence of metabolic engineering employed in the creation and production of "cyanofuels