Optimized genome-wide CRISPR screening enables rapid engineering of growth-based phenotypes in Yarrowia lipolytica.

CRISPR-Cas9 functional genomic screens uncover gene targets linked to various phenotypes for metabolic engineering with remarkable efficiency. However, these genome-wide screens face a number of design challenges, including variable guide RNA activity, ensuring sufficient genome coverage, and maintaining high transformation efficiencies to ensure full library representation. These challenges are prevalent in non-conventional yeast, many of which exhibit traits that are well suited to metabolic engineering and bioprocessing.

A Review on Functionalization of Benzo-5,6-Fused Bicyclic Heteroaromatic Compounds.

Over the decades, functionalized heteroaromatic compounds and their scaffolds have drawn significant attention in synthetic organic chemistry as well as in interdisciplinary sciences due to their prevalence in natural products, hormones, vitamins, and their applications in pharmaceuticals, agrochemicals, veterinary products, dyes and pigments, bio-imaging, semiconductors, and optoelectronics, etc. This review explores various synthetic strategies for functionalization of numerous 5,6 benzo-fused heteroaromatic derivatives such as indole, benzofuran, benzothiophene, benzimidazole, benzoxazole, benzothiazole and benzotriazole through various methods including C-H activation, cross-coupling, metal catalysis, photo-catalysis, electrocatalysis and organocatalysis in recent years (2019-2023). Highly functionalized heterocyclic scaffolds are the important precursors for synthesizing many bioactive drugs like fenbendazole, viozan, griseofulvin, zileuton, flunoxaprofen, natural products, and optoelectronic materials, etc.

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.

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.

Analyzing CRISPR screens in non-conventional microbes.

Unlabelled: 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.

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.

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.

Western Blotting of Membrane-Bound Proteins in Yarrowia lipolytica.

With the discovery of Western blotting as first described by Towbin et al. in 1979, the transfer and visualization of electrophoretically separated proteins on membranes has become the de facto method for the qualitative and quantitative detection of proteins of interest. In this method, proteins are resolved by electrophoresis on a polyacrylamide gel, followed by a transfer of the separated proteins onto a nitrocellulose or polyvinyl difluoride (PVDF) membrane.

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.

Guide RNA Engineering Enables Dual Purpose CRISPR-Cpf1 for Simultaneous Gene Editing and Gene Regulation in .

has fast become a biotechnologically significant yeast for its ability to accumulate lipids to high levels. While there exists a suite of synthetic biology tools for genetic engineering in this yeast, there is a need for multipurposed tools for rapid strain generation. Here, we describe a dual purpose CRISPR-Cpf1 system that is capable of simultaneous gene disruption and gene regulation.

CRISPRi repression of nonhomologous end-joining for enhanced genome engineering via homologous recombination in Yarrowia lipolytica.

In many organisms of biotechnological importance precise genome editing is limited by inherently low homologous recombination (HR) efficiencies. A number of strategies exist to increase the effectiveness of this native DNA repair pathway; however, most strategies rely on permanently disabling competing repair pathways, thus reducing an organism's capacity to repair naturally occurring double strand breaks. Here, we describe a CRISPR interference (CRISPRi) system for gene repression in the oleochemical-producing yeast Yarrowia lipolytica.

Improved ammonium removal from industrial wastewater through systematic adaptation of wild type Chlorella pyrenoidosa.

A single step process is proposed for ammonium removal from nitrogenous industrial effluents, with a concomitant generation of algal biomass. A microalgal strain found in the effluent treatment plant of a fertilizer industry in Mumbai, India was systematically adapted to remove up to 700 ppm of ammoniacal nitrogen from industrial wastewater, which is nearly four times higher than the ammonium tolerance reported in the literature as well as other algal strains tested in our laboratory. 18S rRNA sequencing revealed the strain to be Chlorella pyrenoidosa.