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RNA Binding Proteins As Regulators of Oxidative Stress Identified by a Targeted CRISPR-Cas9 Single Guide RNA Library

ABSTRACT: RNA Binding Proteins As Regulators of Oxidative Stress Identified by a Targeted CRISPR-Cas9 Single Guide RNA Library

PROVIDER: PRJNA769118 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR16220968.fastq.gz	Fastqsanger.gz
	SRR16220969.fastq.gz	Fastqsanger.gz
	SRR16220970.fastq.gz	Fastqsanger.gz
	SRR16220971.fastq.gz	Fastqsanger.gz
	SRR16220972.fastq.gz	Fastqsanger.gz

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RNA Binding Proteins As Regulators of Oxidative Stress Identified by a Targeted CRISPR-Cas9 Single Guide RNA Library

Project description:The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 genome editing system has been broadly adopted for high-throughput genetic screens. However, the application of genome-wide single guide RNA (sgRNA) libraries can be challenging. We generated a custom sgRNA library, an order of magnitude smaller than genome-wide alternatives, to facilitate the genetic screening of RNA binding proteins (RBPs). We demonstrated the utility of our reagent in a genetic screen for RBPs that conveyed cellular resistance or sensitivity to oxidative stress induced by paraquat. This identified that CSDE1 and STRAP, proteins that interact with each other, convey sensitivity to oxidative stress and that Pumilio homologues (PUM1 and PUM2) convey resistance. Targeting eIF4-E1 and -A1 protected cells from high-dose paraquat, whereas eIF4E2 targeted cells did less well. We also found that G3BP1 promoted sensitivity to a low dose of paraquat but protected cells at a higher dose. Our study highlights the use of genetic screens to identify roles of RBPs and identifies novel genes regulating sensitivity to oxidative stress.

2021-10-10 | GSE185462 | GEO

RNA Binding Proteins As Regulators of Oxidative Stress Identified by a Targeted CRISPR-Cas9 Single Guide RNA Library.

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Structure of yeast Argonaute with guide RNA

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Amplicon sequencing of genomically-encoded guide RNA sequences for the Brunello CRISPR library in eHap cells

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2020-04-23 | E-MTAB-8926 | biostudies-arrayexpress

Cas-Database: web-based genome-wide guide RNA library design for gene knockout screens using CRISPR-Cas9.

Project description:CRISPR-derived RNA guided endonucleases (RGENs) have been widely used for both gene knockout and knock-in at the level of single or multiple genes. RGENs are now available for forward genetic screens at genome scale, but single guide RNA (sgRNA) selection at this scale is difficult.We develop an online tool, Cas-Database, a genome-wide gRNA library design tool for Cas9 nucleases from Streptococcus pyogenes (SpCas9). With an easy-to-use web interface, Cas-Database allows users to select optimal target sequences simply by changing the filtering conditions. Furthermore, it provides a powerful way to select multiple optimal target sequences from thousands of genes at once for the creation of a genome-wide library. Cas-Database also provides a web application programming interface (web API) for advanced bioinformatics users.Free access at http://www.rgenome.net/cas-database/sangsubae@hanyang.ac.kr or jskim01@snu.ac.krSupplementary data are available at Bioinformatics online.

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Project description:The development of CRISPR genetic screening tools has improved functional genomics, as these tools enable precise genomic editing, provide broad access to genomic regions beyond protein-coding genes, and have fewer off-target effects than other functional genomics modalities, allowing for novel applications with smaller library sizes compared to prior technologies. Pooled functional genomics screens require high cellular coverage per perturbation to accurately quantify phenotypes and average out phenotype-independent variability across the population. While more compact libraries have decreased the number of cells needed for a given screen, the cell coverage required for large-scale CRISPR screens still poses technical hurdles to screen in more challenging systems, such as iPSC-derived and primary cells. A major factor that influences cell coverage is screening library uniformity, as larger variation in individual guide RNA abundance requires higher cell coverage to reliably measure low-abundance guides. In this work, we have systematically optimized guide RNA cloning procedures to decrease bias. We implement these protocols to demonstrate that CRISPRi screens using 10-fold fewer cells than the current standard provides equivalent statistically significant hit-calling results to screens run at higher coverage, opening the possibility of conducting genome-wide and other large-scale CRISPR screens in technically challenging models.

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Internal guide RNA interactions interfere with Cas9-mediated cleavage.

Project description:The CRISPR/Cas system uses guide RNAs (gRNAs) to direct sequence-specific DNA cleavage. Not every gRNA elicits cleavage and the mechanisms that govern gRNA activity have not been resolved. Low activity could result from either failure to form a functional Cas9-gRNA complex or inability to recognize targets in vivo. Here we show that both phenomena influence Cas9 activity by comparing mutagenesis rates in zebrafish embryos with in vitro cleavage assays. In vivo, our results suggest that genomic factors such as CTCF inhibit mutagenesis. Comparing near-identical gRNA sequences with different in vitro activities reveals that internal gRNA interactions reduce cleavage. Even though gRNAs containing these structures do not yield cleavage-competent complexes, they can compete with active gRNAs for binding to Cas9. These results reveal that both genomic context and internal gRNA interactions can interfere with Cas9-mediated cleavage and illuminate previously uncharacterized features of Cas9-gRNA complex formation.

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