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1.
BMC Genomics ; 22(1): 205, 2021 Mar 23.
Artigo em Inglês | MEDLINE | ID: mdl-33757429

RESUMO

BACKGROUND: 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. RESULTS: 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. CONCLUSIONS: Taken together, we demonstrate a broadly useful platform for comprehensive, high-precision CRISPRi screening in yeast.


Assuntos
Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , RNA Guia de Cinetoplastídeos , Sistemas CRISPR-Cas/genética , Fenótipo , RNA Guia de Cinetoplastídeos/genética , Saccharomyces cerevisiae/genética
2.
Nat Struct Mol Biol ; 30(6): 740-752, 2023 06.
Artigo em Inglês | MEDLINE | ID: mdl-37231154

RESUMO

Numerous proteins regulate gene expression by modulating mRNA translation and decay. To uncover the full scope of these post-transcriptional regulators, we conducted an unbiased survey that quantifies regulatory activity across the budding yeast proteome and delineates the protein domains responsible for these effects. Our approach couples a tethered function assay with quantitative single-cell fluorescence measurements to analyze ~50,000 protein fragments and determine their effects on a tethered mRNA. We characterize hundreds of strong regulators, which are enriched for canonical and unconventional mRNA-binding proteins. Regulatory activity typically maps outside the RNA-binding domains themselves, highlighting a modular architecture that separates mRNA targeting from post-transcriptional regulation. Activity often aligns with intrinsically disordered regions that can interact with other proteins, even in core mRNA translation and degradation factors. Our results thus reveal networks of interacting proteins that control mRNA fate and illuminate the molecular basis for post-transcriptional gene regulation.


Assuntos
Regulação da Expressão Gênica , Proteoma , RNA Mensageiro , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/análise , Proteínas de Saccharomyces cerevisiae/metabolismo , RNA Mensageiro/química , RNA Mensageiro/metabolismo , Processamento Pós-Transcricional do RNA , Estabilidade de RNA , Proteínas de Ligação a RNA/análise , Proteínas de Ligação a RNA/metabolismo
3.
bioRxiv ; 2023 Nov 28.
Artigo em Inglês | MEDLINE | ID: mdl-38076849

RESUMO

The impact of synonymous codon choice on protein output has important implications for understanding endogenous gene expression and design of synthetic mRNAs. Previously, we used a neural network model to design a series of synonymous fluorescent reporters whose protein output in yeast spanned a seven-fold range corresponding to their predicted translation speed. Here, we show that this effect is not due primarily to the established impact of slow elongation on mRNA stability, but rather, that an active mechanism further decreases the number of proteins made per mRNA. We combine simulations and careful experiments on fluorescent reporters to argue that translation initiation is limited on non-optimally encoded transcripts. Using a genome-wide CRISPRi screen to discover factors modulating the output from non-optimal transcripts, we identify a set of translation initiation factors including multiple subunits of eIF3 whose depletion restored protein output of a non-optimal reporter. Our results show that codon usage can directly limit protein production, across the full range of endogenous variability in codon usage, by limiting translation initiation.

4.
Bio Protoc ; 12(7): e4376, 2022 Apr 05.
Artigo em Inglês | MEDLINE | ID: mdl-35530514

RESUMO

Genetic networks regulate nearly all biological processes, including cellular differentiation, homeostasis, and immune responses. Determining the precise role of each gene within a regulatory network can explain its overall, integrated function, and pinpoint mechanisms underlying misregulation in disease states. Transcriptional reporter assays are a useful tool for dissecting these genetic networks, because they link a molecular process to a measurable readout, such as the expression of a fluorescent protein. Here, we introduce a new technique that uses expressed RNA barcodes as reporters, to measure transcriptional changes induced by CRISPRi-mediated genetic perturbation across a diverse, genome-wide library of guide RNAs. We describe an exemplary reporter based on the promoter that drives His4 expression in these guidelines, which can be used as a framework to interrogate other expression phenotypes. In this workflow, a library of plasmids is assembled, encoding a CRISPRi guide RNA (gRNA) along with one or more transcriptional reporters that drive expression of guide-specific nucleotide barcode sequences. For example, when interrogating regulation of the budding yeast HIS4 promoter normalized against a control housekeeping promoter that drives Pgk1 expression, this plasmid library contains a gRNA expression cassette, a HIS4 reporter driving expression of one gRNA-specific nucleotide barcode, and a PGK1 reporter driving expression of a second, gRNA-specific barcode. Long-read sequencing is used to determine which gRNA is associated with these nucleotide barcodes. The plasmid library is then transformed into yeast cells, where each cell receives one plasmid, and experiences a genetic perturbation driven by the guide on that plasmid. The expressed RNA barcodes are extracted in bulk and quantified using high-throughput sequencing, thereby measuring the effect of their corresponding gRNA on barcoded reporter expression. In the case of the HIS4 reporter described above, guides disrupting translation elongation will increase expression of the associated HIS4 barcode specifically, without changing expression of the PGK1 control barcode. It is further possible to quantify plasmid abundance by DNA sequencing, as an additional approach to normalize for differences in plasmid abundance within the population of cells. This protocol outlines the steps to prepare barcode reporter CRISPRi plasmid libraries, link guides to barcodes with long-read sequencing, and measure expression changes through barcode RNA and DNA sequencing. This method is ideal for probing transcriptional or post-transcriptional regulation, as it measures the effects of a genetic perturbation by directly quantifying reporter RNA abundance, rather than relying on indirect growth or fluorescence readouts. Graphic abstract.

5.
FEBS J ; 289(11): 3101-3114, 2022 06.
Artigo em Inglês | MEDLINE | ID: mdl-34914197

RESUMO

DNA damage activates a robust transcriptional stress response, but much less is known about how DNA damage impacts translation. The advent of genome editing with Cas9 has intensified interest in understanding cellular responses to DNA damage. Here, we find that DNA double-strand breaks (DSBs), including those induced by Cas9, trigger the loss of ribosomal protein RPS27A from ribosomes via p53-independent proteasomal degradation. Comparisons of Cas9 and dCas9 ribosome profiling and mRNA-seq experiments reveal a global translational response to DSBs that precedes changes in transcript abundance. Our results demonstrate that even a single DSB can lead to altered translational output and ribosome remodeling, suggesting caution in interpreting cellular phenotypes measured immediately after genome editing.


Assuntos
Quebras de DNA de Cadeia Dupla , Edição de Genes , Sistemas CRISPR-Cas , Dano ao DNA/genética , Reparo do DNA , Edição de Genes/métodos , Proteínas Ribossômicas/genética
6.
Science ; 370(6522)2020 12 11.
Artigo em Inglês | MEDLINE | ID: mdl-33303588

RESUMO

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.


Assuntos
Proteína 9 Associada à CRISPR , Sistemas CRISPR-Cas , Perfilação da Expressão Gênica/métodos , Expressão Gênica , Redes Reguladoras de Genes , Saccharomyces cerevisiae/genética , Oxirredutases do Álcool/genética , Aminoidrolases/genética , Fator de Iniciação 2 em Eucariotos/metabolismo , Fenótipo , Fosforilação , Proteínas Serina-Treonina Quinases/metabolismo , Pirofosfatases/genética , RNA Guia de Cinetoplastídeos/genética , RNA de Transferência/genética , RNA de Transferência/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
7.
ACS Synth Biol ; 8(4): 844-856, 2019 04 19.
Artigo em Inglês | MEDLINE | ID: mdl-30908907

RESUMO

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.


Assuntos
Técnicas de Cultura de Células/métodos , Genômica/métodos , Bactérias/genética , Sítios de Ligação/genética , Genes Essenciais/genética , Genoma/genética , Mutação/genética , Saccharomyces cerevisiae/genética
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