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

ABSTRACT: 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.

SUBMITTER: Turner DJ

PROVIDER: S-EPMC8236562 | biostudies-literature |

REPOSITORIES: biostudies-literature

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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

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

| PRJNA769118 | ENA

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.

| S-EPMC4920116 | biostudies-literature

Genome-scale deletion screening of human long non-coding RNAs using a paired-guide RNA CRISPR-Cas9 library.

Project description:CRISPR-Cas9 screens have been widely adopted to analyze coding-gene functions, but high-throughput screening of non-coding elements using this method is more challenging because indels caused by a single cut in non-coding regions are unlikely to produce a functional knockout. A high-throughput method to produce deletions of non-coding DNA is needed. We report a high-throughput genomic deletion strategy to screen for functional long non-coding RNAs (lncRNAs) that is based on a lentiviral paired-guide RNA (pgRNA) library. Applying our screening method, we identified 51 lncRNAs that can positively or negatively regulate human cancer cell growth. We validated 9 of 51 lncRNA hits using CRISPR-Cas9-mediated genomic deletion, functional rescue, CRISPR activation or inhibition and gene-expression profiling. Our high-throughput pgRNA genome deletion method will enable rapid identification of functional mammalian non-coding elements.

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A Novel Hybrid CNN-SVR for CRISPR/Cas9 Guide RNA Activity Prediction.

Project description:Accurate prediction of guide RNA (gRNA) on-target efficacy is critical for effective application of CRISPR/Cas9 system. Although some machine learning-based and convolutional neural network (CNN)-based methods have been proposed, prediction accuracy remains to be improved. Here, firstly we improved architectures of current CNNs for predicting gRNA on-target efficacy. Secondly, we proposed a novel hybrid system which combines our improved CNN with support vector regression (SVR). This CNN-SVR system is composed of two major components: a merged CNN as the front-end for extracting gRNA feature and an SVR as the back-end for regression and predicting gRNA cleavage efficiency. We demonstrate that CNN-SVR can effectively exploit features interactions from feed-forward directions to learn deeper features of gRNAs and their corresponding epigenetic features. Experiments on commonly used datasets show that our CNN-SVR system outperforms available state-of-the-art methods in terms of prediction accuracy, generalization, and robustness. Source codes are available at https://github.com/Peppags/CNN-SVR.

| S-EPMC6960259 | biostudies-literature

Key sequence features of CRISPR RNA for dual-guide CRISPR-Cas9 ribonucleoprotein complexes assembled with wild-type or HiFi Cas9.

Project description:Specific sequence features of the protospacer and protospacer-adjacent motif (PAM) are critical for efficient cleavage by CRISPR-Cas9, but current knowledge is largely derived from single-guide RNA (sgRNA) systems assessed in cultured cells. In this study, we sought to determine gRNA sequence features of a more native CRISPR-Cas9 ribonucleoprotein (RNP) complex with dual-guide RNAs (dgRNAs) composed of crRNA and tracrRNA, which has been used increasingly in recent CRISPR-Cas9 applications, particularly in zebrafish. Using both wild-type and HiFi SpCas9, we determined on-target cleavage efficiencies of 51 crRNAs in zebrafish embryos by assessing indel occurrence. Statistical analysis of these data identified novel position-specific mononucleotide features relevant to cleavage efficiencies throughout the protospacer sequence that may be unique to CRISPR-Cas9 RNPs pre-assembled with perfectly matched gRNAs. Overall features for wild-type Cas9 resembled those for HiFi Cas9, but specific differences were also observed. Mutational analysis of mononucleotide features confirmed their relevance to cleavage efficiencies. Moreover, the mononucleotide feature-based score, CRISPR-kp, correlated well with efficiencies of gRNAs reported in previous zebrafish RNP injection experiments, as well as independently tested crRNAs only in RNP format, but not with Cas9 mRNA co-injection. These findings will facilitate design of gRNA/crRNAs in genome editing applications, especially when using pre-assembled RNPs.

| S-EPMC8934663 | biostudies-literature

CRISPR library designer (CLD): software for multispecies design of single guide RNA libraries.

Project description:Genetic screens using CRISPR/Cas9 are a powerful method for the functional analysis of genomes.Here we describe CRISPR library designer (CLD), an integrated bioinformatics application for the design of custom single guide RNA (sgRNA) libraries for all organisms with annotated genomes. CLD is suitable for the design of libraries using modified CRISPR enzymes and targeting non-coding regions. To demonstrate its utility, we perform a pooled screen for modulators of the TNF-related apoptosis inducing ligand (TRAIL) pathway using a custom library of 12,471 sgRNAs.CLD predicts a high fraction of functional sgRNAs and is publicly available at https://github.com/boutroslab/cld.

| S-EPMC4807595 | biostudies-literature

Dramatic enhancement of genome editing by CRISPR/Cas9 through improved guide RNA design.

Project description:Success with genome editing by the RNA-programmed nuclease Cas9 has been limited by the inability to predict effective guide RNAs and DNA target sites. Not all guide RNAs have been successful, and even those that were, varied widely in their efficacy. Here we describe and validate a strategy for Caenorhabditis elegans that reliably achieved a high frequency of genome editing for all targets tested in vivo. The key innovation was to design guide RNAs with a GG motif at the 3' end of their target-specific sequences. All guides designed using this simple principle induced a high frequency of targeted mutagenesis via nonhomologous end joining (NHEJ) and a high frequency of precise DNA integration from exogenous DNA templates via homology-directed repair (HDR). Related guide RNAs having the GG motif shifted by only three nucleotides showed severely reduced or no genome editing. We also combined the 3' GG guide improvement with a co-CRISPR/co-conversion approach. For this co-conversion scheme, animals were only screened for genome editing at designated targets if they exhibited a dominant phenotype caused by Cas9-dependent editing of an unrelated target. Combining the two strategies further enhanced the ease of mutant recovery, thereby providing a powerful means to obtain desired genetic changes in an otherwise unaltered genome.

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Efficient CRISPR/Cas9-Mediated Genome Editing Using a Chimeric Single-Guide RNA Molecule.

Project description:The CRISPR/Cas9 system has been applied in diverse eukaryotic organisms for targeted mutagenesis. However, targeted gene editing is inefficient and requires the simultaneous delivery of a DNA template for homology-directed repair (HDR). Here, we used CRISPR/Cas9 to generate targeted double-strand breaks and to deliver an RNA repair template for HDR in rice (Oryza sativa). We used chimeric single-guide RNA (cgRNA) molecules carrying both sequences for target site specificity (to generate the double-strand breaks) and repair template sequences (to direct HDR), flanked by regions of homology to the target. Gene editing was more efficient in rice protoplasts using repair templates complementary to the non-target DNA strand, rather than the target strand. We applied this cgRNA repair method to generate herbicide resistance in rice, which showed that this cgRNA repair method can be used for targeted gene editing in plants. Our findings will facilitate applications in functional genomics and targeted improvement of crop traits.

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Photocontrol of CRISPR/Cas9 function by site-specific chemical modification of guide RNA.

Project description:The function of CRISPR/Cas9 can be conditionally controlled by the rational engineering of guide RNA (gRNA) to target the gene of choice for precise manipulation of the genome. Particularly, chemically modified gRNA that can be activated by using specific stimuli provides a unique tool to expand the versatility of conditional control. Herein, unlike previous engineering of gRNA that generally focused on the RNA part only but neglected RNA-protein interactions, we aimed at the interactive sites between 2'-OH of ribose in the seed region of gRNA and the Cas9 protein and identified that chemical modifications at specific sites could be utilized to regulate the Cas9 activity. By introducing a photolabile group at these specific sites, we achieved optical control of Cas9 activity without disrupting the Watson-Crick base pairing. We further examined our design through CRISPR-mediated gene activation and nuclease cleavage in living cells and successfully manipulated the gene expression by using light irradiation. Our site-specific modification strategy exhibited a highly efficient and dynamic optical response and presented a new perspective for manipulating gRNA based on the RNA-protein interaction rather than the structure of RNA itself. In addition, these specific sites could also be potentially utilized for modification of other stimuli-responsive groups, which would further enrich the toolbox for conditional control of CRISPR/Cas9 function.

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