GMMA Can Stabilize Proteins Across Different Functional Constraints.

Stabilizing proteins without otherwise hampering their function is a central task in protein engineering and design. PYR1 is a plant hormone receptor that has been engineered to bind diverse small molecule ligands. We sought a set of generalized mutations that would provide stability without affecting functionality for PYR1 variants with diverse ligand-binding capabilities. To do this we used a global multi-mutant analysis (GMMA) approach, which can identify substitutions that have stabilizing effects and do not lower function. GMMA has the added benefit of finding substitutions that are stabilizing in different sequence contexts and we hypothesized that applying GMMA to PYR1 with different functionalities would identify this set of generalized mutations. Indeed, conducting FACS and deep sequencing of libraries for PYR1 variants with two different functionalities and applying a GMMA analysis identified 5 substitutions that, when inserted into four PYR1 variants that each bind a unique ligand, provided an increase of 2-6 °C in thermal inactivation temperature and no decrease in functionality.

Repurposing plant hormone receptors as chemically-inducible genetic switches for dynamic regulation in yeast.

Precise control of gene expression is critical for optimizing cellular metabolism and improving the production of valuable biochemicals. However, hard-wired approaches to pathway engineering, such as optimizing promoters, can take time and effort. Moreover, limited tools exist for controlling gene regulation in non-conventional hosts. Here, we develop a two-channel chemically-regulated gene expression system for the multi-stress tolerant yeast Kluyveromyces marxianus and use it to tune ethyl acetate production, a native metabolite produced at high titers in this yeast. To achieve this, we repurposed the plant hormone sensing modules (PYR1ABA/HAB1 and PYR1*MANDI/HAB1*) for high dynamic-range gene activation and repression controlled by either abscisic acid (ABA) or mandipropamid (mandi). To redirect metabolic flux towards ethyl acetate biosynthesis, we simultaneously repress pyruvate dehydrogenase (PDA1) and activate pyruvate decarboxylase (PDC1) to enhance ethyl acetate titers. Thus, we have developed new tools for chemically tuning gene expression in K. marxianus and S. cerevisiae that should be deployable across many non-conventional eukaryotic hosts.

Genome Editing, Transcriptional Regulation, and Forward Genetic Screening Using CRISPR-Cas12a Systems in Yarrowia lipolytica.

Class II Type V endonucleases have increasingly been adapted to develop sophisticated and easily accessible synthetic biology tools for genome editing, transcriptional regulation, and functional genomic screening in a wide range of organisms. One such endonuclease, Cas12a, presents itself as an attractive alternative to Cas9-based systems. The ability to mature its own guide RNAs (gRNAs) from a single transcript has been leveraged for easy multiplexing, and its lack of requirement of a tracrRNA element, also allows for short gRNA expression cassettes. To extend these functionalities into the industrially relevant oleaginous yeast Yarrowia lipolytica, we developed a set of CRISPR-Cas12a vectors for easy multiplexed gene knockout, repression, and activation. We further extended the utility of this CRISPR-Cas12a system to functional genomic screening by constructing a genome-wide guide library targeting every gene with an eightfold coverage. Pooled CRISPR screens conducted with this library were used to profile Cas12a guide activities and develop a machine learning algorithm that could accurately predict highly efficient Cas12a gRNA. In this protocols chapter, we first present a method by which protein coding genes may be functionally disrupted via indel formation with CRISPR-Cas12a systems. Further, we describe how Cas12a fused to a transcriptional regulator can be used in conjunction with shortened gRNA to achieve transcriptional repression or activation. Finally, we describe the design, cloning, and validation of a genome-wide library as well as a protocol for the execution of a pooled CRISPR screen, to determine guide activity profiles in a genome-wide context in Y. lipolytica. The tools and strategies discussed here expand the list of available synthetic biology tools for facile genome engineering in this industrially important host.

An orthogonalized PYR1-based CID module with reprogrammable ligand-binding specificity.

Plants sense abscisic acid (ABA) using chemical-induced dimerization (CID) modules, including the receptor PYR1 and HAB1, a phosphatase inhibited by ligand-activated PYR1. This system is unique because of the relative ease with which ligand recognition can be reprogrammed. To expand the PYR1 system, we designed an orthogonal '*' module, which harbors a dimer interface salt bridge; X-ray crystallographic, biochemical and in vivo analyses confirm its orthogonality. We used this module to create PYR1*MANDI/HAB1* and PYR1*AZIN/HAB1*, which possess nanomolar sensitivities to their activating ligands mandipropamid and azinphos-ethyl. Experiments in Arabidopsis thaliana and Saccharomyces cerevisiae demonstrate the sensitive detection of banned organophosphate contaminants using living biosensors and the construction of multi-input/output genetic circuits. Our new modules enable ligand-programmable multi-channel CID systems for plant and eukaryotic synthetic biology that can empower new plant-based and microbe-based sensing modalities.

High-Performance Cannabinoid Sensor Empowered by Plant Hormone Receptors and Antifouling Magnetic Nanorods.

The misuse of cannabinoids and their synthetic variants poses significant threats to public health, necessitating the development of advanced techniques for detection of these compounds in biological or environmental samples. Existing methods face challenges like lengthy sample pretreatment and laborious antifouling steps. Herein, we present a novel sensing platform using magnetic nanorods coated with zwitterionic polymers for the simple, rapid, and sensitive detection of cannabinoids in biofluids. Our technique utilizes the engineered derivatives of the plant hormone receptor Pyrabactin Resistance 1 (PYR1) as drug recognition elements and employs the chemical-induced dimerization (CID) mechanism for signal development. Additionally, the magnetic nanorods facilitate efficient target capture and reduce the assay duration. Moreover, the zwitterionic polymer coating exhibits excellent antifouling capability, preserving excellent sensor performance in complex biofluids. Our sensors detect cannabinoids in undiluted biofluids like serum, saliva, and urine with a low limit of detection (0.002 pM in saliva and few pM in urine and serum) and dynamic ranges spanning up to 9 orders of magnitude. Moreover, the PYR1 derivatives demonstrate high specificity even in the presence of multiple interfering compounds. This work opens new opportunities for sensor development, showcasing the excellent performance of antifouling magnetic nanorods that can be compatible with different recognition units, including receptors and antibodies, for detecting a variety of targets.

acCRISPR: an activity-correction method for improving the accuracy of CRISPR screens.

High throughput CRISPR screens are revolutionizing the way scientists unravel the genetic underpinnings of engineered and evolved phenotypes. One of the critical challenges in accurately assessing screening outcomes is accounting for the variability in sgRNA cutting efficiency. Poorly active guides targeting genes essential to screening conditions obscure the growth defects that are expected from disrupting them. Here, we develop acCRISPR, an end-to-end pipeline that identifies essential genes in pooled CRISPR screens using sgRNA read counts obtained from next-generation sequencing. acCRISPR uses experimentally determined cutting efficiencies for each guide in the library to provide an activity correction to the screening outcomes via calculation of an optimization metric, thus determining the fitness effect of disrupted genes. CRISPR-Cas9 and -Cas12a screens were carried out in the non-conventional oleaginous yeast Yarrowia lipolytica and acCRISPR was used to determine a high-confidence set of essential genes for growth under glucose, a common carbon source used for the industrial production of oleochemicals. acCRISPR was also used in screens quantifying relative cellular fitness under high salt conditions to identify genes that were related to salt tolerance. Collectively, this work presents an experimental-computational framework for CRISPR-based functional genomics studies that may be expanded to other non-conventional organisms of interest.

Population genomics-guided engineering of phenazine biosynthesis in Pseudomonas chlororaphis.

The emergence of next-generation sequencing (NGS) technologies has made it possible to not only sequence entire genomes, but also identify metabolic engineering targets across the pangenome of a microbial population. This study leverages NGS data as well as existing molecular biology and bioinformatics tools to identify and validate genomic signatures for improving phenazine biosynthesis in Pseudomonas chlororaphis. We sequenced a diverse collection of 34 Pseudomonas isolates using short- and long-read sequencing techniques and assembled whole genomes using the NGS reads. In addition, we assayed three industrially relevant phenotypes (phenazine production, biofilm formation, and growth temperature) for these isolates in two different media conditions. We then provided the whole genomes and phenazine production data to a unitig-based microbial genome-wide association study (mGWAS) tool to identify novel genomic signatures responsible for phenazine production in P. chlororaphis. Post-processing of the mGWAS analysis results yielded 330 significant hits influencing the biosynthesis of one or more phenazine compounds. Based on a quantitative metric (called the phenotype score), we elucidated the most influential hits for phenazine production and experimentally validated them in vivo in the most optimal phenazine producing strain. Two genes significantly increased phenazine-1-carboxamide (PCN) production: a histidine transporter (ProY_1), and a putative carboxypeptidase (PS__04251). A putative MarR-family transcriptional regulator decreased PCN titer when overexpressed in a high PCN producing isolate. Overall, this work seeks to demonstrate the utility of a population genomics approach as an effective strategy in enabling the identification of targets for metabolic engineering of bioproduction hosts.

Analyzing CRISPR screens in non-conventional microbes.

The multifaceted nature of CRISPR screens has propelled advancements in the field of functional genomics. Pooled CRISPR screens involve creating programmed genetic perturbations across multiple genomic sites in a pool of host cells subjected to a challenge, empowering researchers to identify genetic causes of desirable phenotypes. These genome-wide screens have been widely used in mammalian cells to discover biological mechanisms of diseases and drive the development of targeted drugs and therapeutics. Their use in non-model organisms, especially in microbes to improve bioprocessing-relevant phenotypes, has been limited. Further compounding this issue is the lack of bioinformatic algorithms for analyzing microbial screening data with high accuracy. Here, we describe the general approach and underlying principles for conducting pooled CRISPR knockout screens in non-conventional yeasts and performing downstream analysis of the screening data, while also reviewing state-of-the-art algorithms for identification of CRISPR screening outcomes. Application of pooled CRISPR screens to non-model yeasts holds considerable potential to uncover novel metabolic engineering targets and improve industrial bioproduction. ONE-SENTENCE SUMMARY: This mini-review describes experimental and computational approaches for functional genomic screening using CRISPR technologies in non-conventional microbes.

A Closed Form Model for Molecular Ratchet-Type Chemically Induced Dimerization Modules.

Chemical-induced dimerization (CID) modules enable users to implement ligand-controlled cellular and biochemical functions for a number of problems in basic and applied biology. A special class of CID modules occur naturally in plants and involve a hormone receptor that binds a hormone, triggering a conformational change in the receptor that enables recognition by a second binding protein. Two recent reports show that such hormone receptors can be engineered to sense dozens of structurally diverse compounds. As a closed form model for molecular ratchets would be of immense utility in forward engineering of biological systems, here we have developed a closed form model for these distinct CID modules. These modules, which we call molecular ratchets, are distinct from more common CID modules called molecular glues in that they engage in saturable binding kinetics and are characterized well by a Hill equation. A defining characteristic of molecular ratchets is that the sensitivity of the response can be tuned by increasing the molar ratio of the hormone receptor to the binding protein. Thus, the same molecular ratchet can have a pico- or micromolar EC50 depending on the concentration of the different receptor and binding proteins. Closed form models are derived for a base elementary reaction rate model, for ligand-independent complexation of the receptor and binding protein, and for homodimerization of the hormone receptor. Useful governing equations for a variety of in vitro and in vivo applications are derived, including enzyme-linked immunosorbent assay-like microplate assays, transcriptional activation in prokaryotes and eukaryotes, and ligand-induced split protein complementation.

Multiscale simulation-guided design of enzyme bioconjugates with enhanced catalysis.

Biopolymer-scaffold modification is widely used to enhance enzyme catalysis. A central challenge is predicting the effects of scaffold position on enzyme properties. Here, we use a computational-experimental approach to develop a model for the effects of DNA scaffold position on enzyme kinetics. Using phosphotriesterase modified with a 20bp dsDNA, we demonstrate that conjugation position is as important as the scaffold's chemistry and structure. Multiscale simulations predict the effective substrate concentration increases close to the scaffold, which has µM-strength binding to the substrate. Kinetic analysis shows that the effective concentration that the scaffold provides is best utilized when positioned next to, but not blocking, the active site. At ~5Å distance between scaffold and active site a 7-fold increase in k cat /K M was achieved. A model that accounts for the substrate concentration as well PTE-DNA geometry accurately captures the kinetic enhancements, enabling prediction of the effect across a range of DNA positions.

Prospects of thermotolerant Kluyveromyces marxianus for high solids ethanol fermentation of lignocellulosic biomass.

Simultaneous saccharification and fermentation (SSF) is effective for minimizing sugar inhibition during high solids fermentation of biomass solids to ethanol. However, fungal enzymes used during SSF are optimal between 50 and 60 °C, whereas most fermentative yeast, such as Saccharomyces cerevisiae, do not tolerate temperatures above 37 °C. Kluyveromyces marxianus variant CBS 6556 is a thermotolerant eukaryote that thrives at 43 °C, thus potentially serving as a promising new host for SSF operation in biorefineries. Here, we attempt to leverage the thermotolerance of the strain to demonstrate the application of CBS 6556 in a high solids (up to 20 wt% insoluble solid loading) SSF configuration to understand its capabilities and limitations as compared to a proven SSF strain, S. cerevisiae D5A. For this study, we first pretreated hardwood poplar chips using Co-Solvent Enhanced Lignocellulosic Fractionation (CELF) to remove lignin and hemicellulose and to produce cellulose-enriched pretreated solids for SSF. Our results demonstrate that although CBS 6556 could not directly outperform D5A, it demonstrated similar tolerance to high gravity sugar solutions, superior growth rates at higher temperatures and higher early stage ethanol productivity. We discovered that CBS 6556's membrane was particularly sensitive to higher ethanol concentrations causing it to suffer earlier fermentation arrest than D5A. Cross-examination of metabolite data between CBS 6556 and D5A and cell surface imaging suggests that the combined stresses of high ethanol concentrations and temperature to CBS 6556's cell membrane was a primary factor limiting its ethanol productivity. Hence, we believe K. marxianus to be an excellent host for future genetic engineering efforts to improve membrane robustness especially at high temperatures in order to achieve higher ethanol productivity and titers, serving as a viable alternative to D5A.

RNA polymerase II-driven CRISPR-Cas9 system for efficient non-growth-biased metabolic engineering of Kluyveromyces marxianus.

The thermotolerant yeast Kluyveromyces marxianus has gained significant attention in recent years as a promising microbial candidate for industrial biomanufacturing. Despite several contributions to the expanding molecular toolbox for gene expression and metabolic engineering of K. marxianus, there remains a need for a more efficient and versatile genome editing platform. To address this, we developed a CRISPR-based editing system that enables high efficiency marker-less gene disruptions and integrations using only 40 bp homology arms in NHEJ functional and non-functional K. marxianus strains. The use of a strong RNA polymerase II promoter allows efficient expression of gRNAs flanked by the self-cleaving RNA structures, tRNA and HDV ribozyme, from a single plasmid co-expressing a codon optimized Cas9. Implementing this system resulted in nearly 100% efficiency of gene disruptions in both NHEJ-functional and NHEJ-deficient K. marxianus strains, with donor integration efficiencies reaching 50% and 100% in the two strains, respectively. The high gRNA targeting performance also proved instrumental for selection of engineered strains with lower growth rate but improved polyketide biosynthesis by avoiding an extended outgrowth period, a common method used to enrich for edited cells but that fails to recover advantageous mutants with even slightly impaired fitness. Finally, we provide the first demonstration of simultaneous, markerless integrations at multiple loci in K. marxianus using a 2.6 kb and a 7.6 kb donor, achieving a dual integration efficiency of 25.5% in a NHEJ-deficient strain. These results highlight both the ease of use and general robustness of this system for rapid and flexible metabolic engineering in this non-conventional yeast.

A reverse-selective ion exchange membrane for the selective transport of phosphates via an outer-sphere complexation-diffusion pathway.

Specific-ion selectivity is a highly desirable feature for the next generation of membranes. However, existing membranes rely on differences in charge, size and hydration energy, which limits their ability to target individual ion species. Here we demonstrate a nanocomposite ion-exchange membrane material that enables a reverse-selective transport mechanism that can selectively pass a single ion species. We demonstrate this transport mechanism with phosphate ions selectively transporting across negatively charged cation exchange membranes. Selective transport is enabled by the in situ growth of hydrous manganese oxide nanoparticles throughout a cation exchange membrane that provide a diffusion pathway via phosphate-specific, reversible outer-sphere interactions. On incorporating the hydrous manganese oxide nanoparticles, the membrane's phosphate flux increased by a factor of 27 over an unmodified cation exchange membrane, and the selectivity of phosphorous over sulfate, nitrate and chloride reaches 47, 100 and 20, respectively. By pairing ion-specific outer-sphere interactions between the target ions and appropriate nanoparticles, these nanocomposite ion-exchange materials can, in principle, achieve selective transport for a range of ions.

Genome-wide CRISPR-Cas9 screen reveals a persistent null-hyphal phenotype that maintains high carotenoid production in Yarrowia lipolytica.

Yarrowia lipolytica is a metabolic engineering host of growing industrial interest due to its ability to metabolize hydrocarbons, fatty acids, glycerol, and other renewable carbon sources. This dimorphic yeast undergoes a stress-induced transition to a multicellular hyphal state, which can negatively impact biosynthetic activity, reduce oxygen and nutrient mass transfer in cell cultures, and increase culture viscosity. Identifying mutations that prevent the formation of hyphae would help alleviate the bioprocess challenges that they create. To this end, we conducted a genome-wide CRISPR screen to identify genetic knockouts that prevent the transition to hyphal morphology. The screen identified five mutants with a null-hyphal phenotype-ΔRAS2, ΔRHO5, ΔSFL1, ΔSNF2, and ΔPAXIP1. Of these hits, only ΔRAS2 suppressed hyphal formation in an engineered lycopene production strain over a multiday culture. The RAS2 knockout was also the only genetic disruption characterized that did not affect lycopene production, producing more than 5 mg L-1 OD-1 from a heterologous pathway with enhanced carbon flux through the mevalonate pathway. These data suggest that a ΔRAS2 mutant of Y. lipolytica could prove useful in engineering a metabolic engineering host of the production of carotenoids and other biochemicals.

Rapid biosensor development using plant hormone receptors as reprogrammable scaffolds.

A general method to generate biosensors for user-defined molecules could provide detection tools for a wide range of biological applications. Here, we describe an approach for the rapid engineering of biosensors using PYR1 (Pyrabactin Resistance 1), a plant abscisic acid (ABA) receptor with a malleable ligand-binding pocket and a requirement for ligand-induced heterodimerization, which facilitates the construction of sense-response functions. We applied this platform to evolve 21 sensors with nanomolar to micromolar sensitivities for a range of small molecules, including structurally diverse natural and synthetic cannabinoids and several organophosphates. X-ray crystallography analysis revealed the mechanistic basis for new ligand recognition by an evolved cannabinoid receptor. We demonstrate that PYR1-derived receptors are readily ported to various ligand-responsive outputs, including enzyme-linked immunosorbent assay (ELISA)-like assays, luminescence by protein-fragment complementation and transcriptional circuits, all with picomolar to nanomolar sensitivity. PYR1 provides a scaffold for rapidly evolving new biosensors for diverse sense-response applications.

Genome-wide functional screens enable the prediction of high activity CRISPR-Cas9 and -Cas12a guides in Yarrowia lipolytica.

Genome-wide functional genetic screens have been successful in discovering genotype-phenotype relationships and in engineering new phenotypes. While broadly applied in mammalian cell lines and in E. coli, use in non-conventional microorganisms has been limited, in part, due to the inability to accurately design high activity CRISPR guides in such species. Here, we develop an experimental-computational approach to sgRNA design that is specific to an organism of choice, in this case the oleaginous yeast Yarrowia lipolytica. A negative selection screen in the absence of non-homologous end-joining, the dominant DNA repair mechanism, was used to generate single guide RNA (sgRNA) activity profiles for both SpCas9 and LbCas12a. This genome-wide data served as input to a deep learning algorithm, DeepGuide, that is able to accurately predict guide activity. DeepGuide uses unsupervised learning to obtain a compressed representation of the genome, followed by supervised learning to map sgRNA sequence, genomic context, and epigenetic features with guide activity. Experimental validation, both genome-wide and with a subset of selected genes, confirms DeepGuide's ability to accurately predict high activity sgRNAs. DeepGuide provides an organism specific predictor of CRISPR guide activity that with retraining could be applied to other fungal species, prokaryotes, and other non-conventional organisms.

Systemic immune responses to irradiated tumours via the transport of antigens to the tumour periphery by injected flagellate bacteria.

Because the tumour microenvironment is typically immunosuppressive, the release of tumour antigens mediated by radiotherapy or chemotherapy does not sufficiently activate immune responses. Here we show that, following radiotherapy, the intratumoural injection of a genetically attenuated strain of Salmonella coated with antigen-adsorbing cationic polymer nanoparticles caused the accumulation of tumour antigens at the tumour's periphery. This enhanced the crosstalk between the antigens and dendritic cells, and resulted in large increases in activated ovalbumin-specific dendritic cells in vitro and in systemic antitumour effects, and extended survival in multiple tumour models in mice, including a model of metastasis and recurrence. The antitumour effects were abrogated by the antibody-mediated depletion of CD8+ T cells, indicating that systemic tumour regression was caused by adaptive immune responses. Leveraging flagellate bacteria to transport tumour antigens to the periphery of tumours to potentiate the activation of dendritic cells may open up new strategies for in situ cancer vaccination.

Microenvironmental effects can masquerade as substrate channelling in cascade biocatalysis.

Natural cascades frequently use spatial organization to introduce beneficial substrate channeling mechanisms, a strategy that has been widely mimicked in many engineered multienzyme cascades with enhanced catalysis. Enabled by new molecular scaffolds it is now possible to test the effects of spatial organization on cascade kinetics; however, these scaffolds can also alter the microenvironment experienced by the assembled enzymes. We know from decades of enzyme immobilization research that the microenvironment affects enzymatic activity, thus complicating kinetic analysis. Here, we review these effects and discuss examples that exploit the microenvironment to improve single enzyme and cascade catalysis. In doing so, we highlight the challenges in ascribing kinetic enhancements directly to substrate channeling without first determining the effects of the microenvironment.

Hydrolysis of lignocellulose by anaerobic fungi produces free sugars and organic acids for two-stage fine chemical production with Kluyveromyces marxianus.

Development of the bioeconomy is driven by our ability to access the energy-rich carbon trapped in recalcitrant plant materials. Current strategies to release this carbon rely on expensive enzyme cocktails and physicochemical pretreatment, producing inhibitory compounds that hinder subsequent microbial bioproduction. Anaerobic fungi are an appealing solution as they hydrolyze crude, untreated biomass at ambient conditions into sugars that can be converted into value-added products by partner organisms. However, some carbon is lost to anaerobic fungal fermentation products. To improve efficiency and recapture this lost carbon, we built a two-stage bioprocessing system pairing the anaerobic fungus Piromyces indianae with the yeast Kluyveromyces marxianus, which grows on a wide range of sugars and fermentation products. In doing so we produce fine and commodity chemicals directly from untreated lignocellulose. P. indianae efficiently hydrolyzed substrates such as corn stover and poplar to generate sugars, fermentation acids, and ethanol, which K. marxianus consumed while producing 2.4 g/L ethyl acetate. An engineered strain of K. marxianus was also able to produce 550 mg/L 2-phenylethanol and 150 mg/L isoamyl alcohol from P. indianae hydrolyzed lignocellulosic biomass. Despite the use of crude untreated plant material, production yields were comparable to optimized rich yeast media due to the use of all available carbon including organic acids, which formed up to 97% of free carbon in the fungal hydrolysate. This work demonstrates that anaerobic fungal pretreatment of lignocellulose can sustain the production of fine chemicals at high efficiency by partnering organisms with broad substrate versatility.

Guide RNA Design for Genome-Wide CRISPR Screens in Yarrowia lipolytica.

Genome-wide functional genomic screens are essential to determining the genetic underpinning of a biological process. Novel and powerful tools for perturbing gene function, with the help of genetic and epigenetic information, have made it possible to systematically investigate the contribution of every gene to evolved and engineered phenotypes. Functional genomics and screening for enhanced phenotypes become ever more important when dealing with nonconventional hosts. Non-model organisms are valuable to metabolic engineering as they present a range of desirable phenotypes and can help in avoiding complex and intensive engineering of less suitable hosts that do not possess the desired phenotype(s). Domestication of such hosts however requires a suite of synthetic biology tools that allow for targeted genome engineering, regulation of gene expression, and genome-wide mutational screens. The widespread adoption of CRISPR-Cas9 and CRISPR-Cpf1 based systems has allowed for such screens in many organisms. Key considerations in any genome-wide CRISPR screen are the design of a set of unique guide RNAs targeting the required set of genes in the genome and the design of nontargeting guide RNAs that function as appropriate negative controls for the experiment. In this methods chapter, we present protocols for the design of guides for a CRISPR screen, targeting every gene in the genome of the industrially relevant oleaginous yeast Yarrowia lipolytica. The first set of protocols describes the algorithm for the design of genome targeting and nontargeting guides for a genome-wide CRISPR-Cpf1 screen. The second set of protocols describes modifications to the first for the design of guides for a CRISPR-Cas9 screen. The strategies described here should serve as an efficient guide to design a library of gRNAs for most genome-wide CRISPR screens.