Zuriah A. Meacham

Bio: Zuriah A. Meacham is an academic researcher from University of California, Berkeley. The author has contributed to research in topics: Cas9 & CRISPR interference. The author has an hindex of 5, co-authored 8 publications receiving 55 citations.

An Accessible Continuous-Culture Turbidostat for Pooled Analysis of Complex Libraries

Abstract: We present an accessible, robust continuous-culture turbidostat system that greatly facilitates the generation and phenotypic analysis of highly complex libraries in yeast and bacteria. Our system has many applications in genomics and systems biology; here, we demonstrate three of these uses. We first measure how the growth rate of budding yeast responds to limiting nitrogen at steady state and in a dynamically varying environment. We also demonstrate the direct selection of a diverse, genome-scale protein fusion library in liquid culture. Finally, we perform a comprehensive mutational analysis of the essential gene RPL28 in budding yeast, mapping sequence constraints on its wild-type function and delineating the binding site of the drug cycloheximide through resistance mutations. Our system can be constructed and operated with no specialized skills or equipment and applied to study genome-wide mutant pools and diverse libraries of sequence variants under well-defined growth conditions.

A genome-scale CRISPR interference guide library enables comprehensive phenotypic profiling in yeast

Abstract: CRISPR/Cas9-mediated transcriptional interference (CRISPRi) enables programmable gene knock-down, yielding loss-of-function phenotypes for nearly any gene. Effective, inducible CRISPRi has been demonstrated in budding yeast, and genome-scale guide libraries enable systematic, genome-wide genetic analysis. We present a comprehensive yeast CRISPRi library, based on empirical design rules, containing 10 distinct guides for most genes. Competitive growth after pooled transformation revealed strong fitness defects for most essential genes, verifying that the library provides comprehensive genome coverage. We used the relative growth defects caused by different guides targeting essential genes to further refine yeast CRISPRi design rules. In order to obtain more accurate and robust guide abundance measurements in pooled screens, we link guides with random nucleotide barcodes and carry out linear amplification by in vitro transcription. Taken together, we demonstrate a broadly useful platform for comprehensive, high-precision CRISPRi screening in yeast.

CiBER-seq dissects genetic networks by quantitative CRISPRi profiling of expression phenotypes.

Abstract: To realize the promise of CRISPR-Cas9-based genetics, approaches are needed to quantify a specific, molecular phenotype across genome-wide libraries of genetic perturbations. We addressed this challenge by profiling transcriptional, translational, and posttranslational reporters using CRISPR interference (CRISPRi) with barcoded expression reporter sequencing (CiBER-seq). Our barcoding approach allowed us to connect an entire library of guides to their individual phenotypic consequences using pooled sequencing. CiBER-seq profiling fully recapitulated the integrated stress response (ISR) pathway in yeast. Genetic perturbations causing uncharged transfer RNA (tRNA) accumulation activated ISR reporter transcription. Notably, tRNA insufficiency also activated the reporter, independent of the uncharged tRNA sensor. By uncovering alternate triggers for ISR activation, we illustrate how precise, comprehensive CiBER-seq profiling provides a powerful and broadly applicable tool for dissecting genetic networks.

A genome-scale CRISPR interference guide library enables comprehensive phenotypic profiling in yeast

Abstract: CRISPR/Cas9-mediated transcriptional interference (CRISPRi) enables programmable gene knock-down, yielding interpretable loss-of-function phenotypes for nearly any gene. Effective, inducible CRISPRi has been demonstrated in budding yeast, but no genome-scale guide libraries have been reported. We present a comprehensive yeast CRISPRi library, based on empirical design rules, containing 10 distinct guides for most genes. Competitive growth after pooled transformation revealed strong fitness defects for most essential genes, verifying that the library provides comprehensive genome coverage. We used the relative growth defects caused by different guides targeting essential genes to further refine yeast CRISPRi design rules. In order to obtain more accurate and robust guide abundance measurements in pooled screens, we link guides with random nucleotide barcodes and carry out linear amplification by in vitro transcription. Taken together, we demonstrate a broadly useful platform for comprehensive, high-precision CRISPRi screening in yeast.

CiBER-seq dissects genetic networks by quantitative CRISPRi profiling of expression phenotypes

Abstract: To realize the promise of CRISPR/Cas9-based genetics, approaches are needed to quantify a specific, molecular phenotype across genome-wide libraries of genetic perturbations. We address this challenge by profiling transcriptional, translational, and post-translational reporters using CRISPR interference with barcoded expression reporter sequencing (CiBER-seq). Our barcoding approach connects an entire library of guides to their individual phenotypic consequences using pooled sequencing. We show that CiBER-seq profiling fully recapitulates the integrated stress response (ISR) pathway in yeast. Genetic perturbations causing uncharged tRNA accumulation activated ISR reporter transcription. Surprisingly, tRNA insufficiency also activated the reporter, independent of the Gcn2 kinase that senses uncharged tRNAs. By uncovering alternate triggers for ISR activation, we illustrate how precise, comprehensive CiBER-seq profiling provides a powerful and broadly applicable tool for dissecting genetic networks.

A Multiplexed Single-Cell CRISPR Screening Platform Enables Systematic Dissection of the Unfolded Protein Response

Abstract: Functional genomics efforts face tradeoffs between number of perturbations examined and complexity of phenotypes measured. We bridge this gap with Perturb-seq, which combines droplet-based single-cell RNA-seq with a strategy for barcoding CRISPR-mediated perturbations, allowing many perturbations to be profiled in pooled format. We applied Perturb-seq to dissect the mammalian unfolded protein response (UPR) using single and combinatorial CRISPR perturbations. Two genome-scale CRISPR interference (CRISPRi) screens identified genes whose repression perturbs ER homeostasis. Subjecting ∼100 hits to Perturb-seq enabled high-precision functional clustering of genes. Single-cell analyses decoupled the three UPR branches, revealed bifurcated UPR branch activation among cells subject to the same perturbation, and uncovered differential activation of the branches across hits, including an isolated feedback loop between the translocon and IRE1α. These studies provide insight into how the three sensors of ER homeostasis monitor distinct types of stress and highlight the ability of Perturb-seq to dissect complex cellular responses.

Perturb-Seq: Dissecting Molecular Circuits with Scalable Single-Cell RNA Profiling of Pooled Genetic Screens

Abstract: Genetic screens help infer gene function in mammalian cells, but it has remained difficult to assay complex phenotypes-such as transcriptional profiles-at scale. Here, we develop Perturb-seq, combining single-cell RNA sequencing (RNA-seq) and clustered regularly interspaced short palindromic repeats (CRISPR)-based perturbations to perform many such assays in a pool. We demonstrate Perturb-seq by analyzing 200,000 cells in immune cells and cell lines, focusing on transcription factors regulating the response of dendritic cells to lipopolysaccharide (LPS). Perturb-seq accurately identifies individual gene targets, gene signatures, and cell states affected by individual perturbations and their genetic interactions. We posit new functions for regulators of differentiation, the anti-viral response, and mitochondrial function during immune activation. By decomposing many high content measurements into the effects of perturbations, their interactions, and diverse cell metadata, Perturb-seq dramatically increases the scope of pooled genomic assays.

The helicase Ded1p controls use of near-cognate translation initiation codons in 5′ UTRs

Abstract: The conserved and essential DEAD-box RNA helicase Ded1p from yeast and its mammalian orthologue DDX3 are critical for the initiation of translation1. Mutations in DDX3 are linked to tumorigenesis2-4 and intellectual disability5, and the enzyme is targeted by a range of viruses6. How Ded1p and its orthologues engage RNAs during the initiation of translation is unknown. Here we show, by integrating transcriptome-wide analyses of translation, RNA structure and Ded1p-RNA binding, that the effects of Ded1p on the initiation of translation are connected to near-cognate initiation codons in 5' untranslated regions. Ded1p associates with the translation pre-initiation complex at the mRNA entry channel and repressing the activity of Ded1p leads to the accumulation of RNA structure in 5' untranslated regions, the initiation of translation from near-cognate start codons immediately upstream of these structures and decreased protein synthesis from the corresponding main open reading frames. The data reveal a program for the regulation of translation that links Ded1p, the activation of near-cognate start codons and mRNA structure. This program has a role in meiosis, in which a marked decrease in the levels of Ded1p is accompanied by the activation of the alternative translation initiation sites that are seen when the activity of Ded1p is repressed. Our observations indicate that Ded1p affects translation initiation by controlling the use of near-cognate initiation codons that are proximal to mRNA structure in 5' untranslated regions.

Continuous Cultivation as a Tool Toward the Rational Bioprocess Development With Pichia Pastoris Cell Factory.

Abstract: The methylotrophic yeast Pichia pastoris (Komagataella phaffii) is currently considered one of the most promising hosts for recombinant protein production (RPP) and metabolites due to the availability of several tools to efficiently regulate the recombinant expression, its ability to perform eukaryotic post-translational modifications and to secrete the product in the extracellular media. The challenge of improving the bioprocess efficiency can be faced from two main approaches: the strain engineering, which includes enhancements in the recombinant expression regulation as well as overcoming potential cell capacity bottlenecks; and the bioprocess engineering, focused on the development of rational-based efficient operational strategies. Understanding the effect of strain and operational improvements in bioprocess efficiency requires to attain a robust knowledge about the metabolic and physiological changes triggered into the cells. For this purpose, a number of studies have revealed chemostat cultures to provide a robust tool for accurate, reliable, and reproducible bioprocess characterization. It should involve the determination of key specific rates, productivities, and yields for different C and N sources, as well as optimizing media formulation and operating conditions. Furthermore, studies along the different levels of systems biology are usually performed also in chemostat cultures. Transcriptomic, proteomic and metabolic flux analysis, using different techniques like differential target gene expression, protein description and 13C-based metabolic flux analysis, are widely described as valued examples in the literature. In this scenario, the main advantage of a continuous operation relies on the quality of the homogeneous samples obtained under steady-state conditions, where both the metabolic and physiological status of the cells remain unaltered in an all-encompassing picture of the cell environment. This contribution aims to provide the state of the art of the different approaches that allow the design of rational strain and bioprocess engineering improvements in Pichia pastoris toward optimizing bioprocesses based on the results obtained in chemostat cultures. Interestingly, continuous cultivation is also currently emerging as an alternative operational mode in industrial biotechnology for implementing continuous process operations.

An inducible CRISPR interference library for genetic interrogation of Saccharomyces cerevisiae biology.

Abstract: Genome-scale CRISPR interference (CRISPRi) is widely utilized to study cellular processes in a variety of organisms. Despite the dominance of Saccharomyces cerevisiae as a model eukaryote, an inducible genome-wide CRISPRi library in yeast has not yet been presented. Here, we present a genome-wide, inducible CRISPRi library, based on spacer design rules optimized for S. cerevisiae. We have validated this library for genome-wide interrogation of gene function across a variety of applications, including accurate discovery of haploinsufficient genes and identification of enzymatic and regulatory genes involved in adenine and arginine biosynthesis. The comprehensive nature of the library also revealed refined spacer design parameters for transcriptional repression, including location, nucleosome occupancy and nucleotide features. CRISPRi screens using this library can identify genes and pathways with high precision and a low false discovery rate across a variety of experimental conditions, enabling rapid and reliable assessment of genetic function and interactions in S. cerevisiae.