Plasmid and Sequencing Library Preparation for CRISPRi Barcoded Expression Reporter Sequencing (CiBER-seq) in Saccharomyces cerevisiae .

Autor:	Muller RY; Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, USA., Meacham ZA; Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, USA., Ingolia NT; Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, USA.; California Institute for Quantitative Biosciences, University of California, Berkeley, Berkeley, USA.
Jazyk:	angličtina
Zdroj:	Bio-protocol [Bio Protoc] 2022 Apr 05; Vol. 12 (7), pp. e4376. Date of Electronic Publication: 2022 Apr 05 (Print Publication: 2022).
DOI:	10.21769/BioProtoc.4376
Abstrakt:	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. Competing Interests: Competing interestsThe authors report no competing financial or non-financial interests. (Copyright © 2022 The Authors; exclusive licensee Bio-protocol LLC.)
Databáze:	MEDLINE
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