Biology subjects

Tafrishi, A.

Publications and source records attributed to Tafrishi, A..

5 recordsLinked to original sources

bioRxiv2025

Kingdom-Wide CRISPR Guide Design with ALLEGRO

Designing CRISPR single guide RNA (sgRNA) libraries targeting entire kingdoms of life will significantly advance genetic research in diverse and underexplored taxa. Current sgRNA design tools are often species-specific and fail to scale to large, phylogenetically diverse datasets, limiting their applicability to comparative genomics, evolutionary studies, and biotechnology. Here, we present ALLEGRO, a combinatorial optimization algorithm able to design minimal, yet highly effective sgRNA libraries targeting thousands of species. Leveraging integer linear programming, ALLEGRO identified compact sgRNA sets simultaneously targeting several genes of interest for over 2,000 species across the fungal kingdom. We experimentally validated the sgRNAs designed by ALLEGRO in Kluyveromyces marxianus, Komagataella phaffii, and Yarrowia lipolytica. In addition, we adopted a generalized Cas9-Ribonucleoprotein delivery system coupled with protoplast transformation to extend ALLEGROs sgRNA libraries to other untested fungal genomes, such as Rhodotorula araucariae. Our experimental results, along with cross-validation, show that ALLEGRO enables efficient CRISPR genome editing, supporting the development of universal sgRNA libraries applicable to entire taxonomic groups.

bioinformatics↗

bioRxiv2025

Ribo-seq guided design of enhanced protein secretion in Komagataella phaffii

The production of recombinant proteins requires the precise coordination of various biological processes, including protein synthesis, folding, trafficking, and secretion. The overproduction of a heterologous protein can impose various bottlenecks on these networks. Identifying and alleviating these bottlenecks can guide strain engineering efforts to enhance protein production. The methylotrophic yeast Komagataella phaffii is used for its high capacity to produce recombinant proteins. Here, we use ribosome profiling to identify bottlenecks in protein secretion during heterologous expression of human serum albumin (HSA). Validation of this analysis showed that the knockout of non-essential genes whose gene products target the ER, through co- and post-translational mechanisms, and have high ribosome utilization can increase production of a heterologous protein, HSA. A triple knockout in co-translationally translocated carbohydrate and acetate transporter Gal2p, cell wall maintenance protein Ydr134cp, and the post-translationally translocated cell wall protein Aoa65896.1 increased HSA production by 35%. This data-driven strain engineering approach uses cell-level information to identify gene targets for phenotype improvement. This specific case identifies hits and creates strains with improved HSA production, with Ribo-seq and bioinformatic analysis to identify non-essential ER targeted proteins that are high ribosome utilizers.

systems biology↗

bioRxiv2024

PYR1 biosensor-driven genome-wide CRISPR screens for improved monoterpenoid production in Kluyveromyces marxianus

Monoterpenoids are valued for their roles as flavors, fragrances, insecticides, and energy-dense fuels. Microbial biosynthesis offers sustainable biosynthesis routes for these important molecules, but production levels remain limited. Here, we introduce a biosensor-driven microbial engineering strategy to enhance monoterpenoid production, specifically targeting geraniol. Using mutagenized libraries of the PYR1 receptor--a versatile biosensor from plant ABA signaling pathways with a malleable binding pocket--we screened 24 monoterpenoids and identified PYR1 variants responsive to eight, including geraniol. A low background, highly selective geraniol-sensitive PYR1 variant was expressed in the thermotolerant yeast Kluyveromyces marxianus as a growth-based biosensor circuit, allowing for rapid strain engineering. By coupling the geraniol-sensitive PYR1 sensor with a genome-wide CRISPR-Cas9 mutagenesis approach, we identified six gene knockouts that enhance geraniol production, achieving up to a 2-fold increase in titer. This study demonstrates the power of the PYR1 biosensor platform to enable rapid strain engineering and the identification of mutants that improve titer of a desired metabolite.

synthetic biology↗

bioRxiv2024

Functional genomic screening in Komagataella phaffii enabled by high-activity CRISPR-Cas9 library

CRISPR-based high-throughput genome-wide loss-of-function screens are a valuable approach to functional genetics and strain engineering. The yeast Komagataella phaffii is a host of particular interest in the biopharmaceutical industry and as a metabolic engineering host for proteins and metabolites. Here, we design and validate a highly active 6-fold coverage genome-wide sgRNA library for this biotechnologically important yeast containing 30,848 active sgRNAs targeting over 99% of its coding sequences. Conducting fitness screens in the absence of functional non-homologous end joining (NHEJ), the dominant DNA repair mechanism in K. phaffii, provides a quantitative means to assess the activity of each sgRNA in the library. This approach allows for the experimental validation of each guides targeting activity, leading to more precise screening outcomes. We used this approach to conduct growth screens with glucose as the sole carbon source and identify essential genes. Comparative analysis of the called gene sets identified a core set of K. phaffii essential genes, many of which relate to protein production, secretion, and glycosylation. The high activity, genome-wide CRISPR library developed here enables functional genomic screening in K. phaffii, applied here to gene essentiality classification, and promises to enable other genetic screens. HighlightsO_LIDesigned and validated a high activity genome-wide CRISPR-Cas9 library for K. phaffii C_LIO_LIDisabling NHEJ DNA repair enables the generation of genome-wide guide activity profiles C_LIO_LIActivity-corrected fitness screens identify a high confidence set of essential genes in K. phaffii C_LIO_LIProtein production, secretion, and glycosylation pathways are essential in K. phaffii but not in other yeasts C_LI

systems biology↗

bioRxiv2022

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 novel 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, thus determining the fitness effect of disrupted genes. This is accomplished by calculating an optimization metric that quantifies the tradeoff between guide activity and library coverage, which is maximized to accurately classify genes essential to screening conditions. CRISPR-Cas9 and -Cas12a screens were carried out in the non-conventional oleaginous yeast Yarrowia lipolytica 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 gain-and loss-of-function screens under high salt and low pH conditions to identify known and novel genes that were related to stress 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.

synthetic biology↗