Systematic Evaluation of crRNA Design Parameters for Optimized Cas13d-Mediated RNA Targeting in Chicken Cells
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PROVIDER: PRJNA1307436 | ENA |
REPOSITORIES: ENA
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Project description:CRISPR/Cas13 systems are increasingly becoming popular tools for programmable RNA targeting owing to its high-precision and ease at which crRNAs can be designed against mRNA targets of interest, often surpassing the usability of traditional RNA-targeting tools such as RNAi. The RNA-targeting capabilities of CRISPR/Cas13 have been harnessed to correct pathogenic mutations and combat infectious diseases. However, the wider adoption of Cas13 systems relies on the ability to design effective crRNAs that are broadly targetable and resistant to emerging mutants. In this study, we delineated the principles for the development of effective crRNAs by targeting DsRed fluorescence reporter and synthetic influenza mRNA in chicken fibroblast DF1 cells. To systematically determine the optimal design for crRNAs, we designed multiple versions of crRNAs to investigate the minimum length of the crRNA, importance of protospacer flanking sequence, degree of mismatch tolerance, and off target effects. Our data revealed variable knockdown levels between crRNAs, in which a few crRNAs achieved over 95% target knockdown. We showed that crRNAs exhibit a high degree of tolerance to single-nucleotide mismatches, regardless of the position at which the single-nt mismatch was introduced. However, 4-nt mismatches within crRNAs significantly reduced targeting efficacy, and eight nucleotide mismatches completely diminished targeting efficacy. Finally, we compared targeting efficiency of two widely used Cas13d variants (RfxCas13d and HfCas13d) and associated collateral degradation of bystander RNA. Our data extend current understanding of Cas13d-mediated RNA targeting and offer a framework for rational crRNA design to enhance effectiveness in diverse applications, including antiviral strategies.
2025-08-26 | GSE305666 | GEO
Project description:Type VI CRISPR enzymes are RNA-targeting proteins with nuclease activity that enable specific and robust target gene knockdown without altering the genome. To define rules for the design of Cas13d guide RNAs, we conducted massively parallel screens targeting messenger RNAs of a green fluorescent protein transgene, and CD46, CD55 and CD71 cell-surface proteins in human cells. In total, we measured the activity of 24,460 gRNAs with and without mismatches relative to the target sequences. Knockdown efficacy is driven by gRNA-specific features and target site context. Single mismatches generally reduce knockdown to a modest degree, but spacer nucleotides 15–21 are largely intolerant of target site mismatches. We developed a computational model to identify optimal gRNAs and confirm their generalizability testing 3,979 guides targeting mRNAs of 48 endogenous genes. We show that Cas13 can be used in forward transcriptomic pooled screens and, using our model, predict optimized Cas13 gRNAs for all protein-coding transcripts in the human genome.
2020-01-30 | GSE142675 | GEO
Project description:Massively parallel Cas13d screens reveal principles for guide RNA design
| PRJNA597906 | ENA
Project description:We performed an experimental Cas13d-SARScov2 genome-wide screen to identify gRNAs that would allow Cas13d to degrade the viral RNA. We built mCherry reporter plasmids that express mCherry with a 3kb 3'UTR deriving from the SARScov2 genome. In total we designed 11 reporters covering the entire plus strand of the viral genome and 11 other reporters covering the entire minus strand. Each of the 22 mCherry reporter plasmids carries a U6 expression cassette containing a Cas13d gRNA that targets the 3'UTR of the mCherry reporter. Each reporter is represented by a pool of reporters each containing a different gRNA that targets mCherry 3'UTR for a total average of ~300 gRNA per 3'UTR. The entire pool of 22 reporters, each with a pool of ~300 different gRNAs constitutes a comprehensive set ~6,500 reporters (~ 6,500 different gRNAs) that allowed us to interrogate the entire SARScov2 plus and minus strand viral RNA for regions of vulnerability and targetability. In order to specifically interrogate Cas13d activity an remove the biases that would be introduced in the reporter expression by the presence of a large 3kb 3'UTR we used a case (presence of Cas13d) control (absence of Cas13d) design. Briefly, the ~6,500 reporters were lentiviral transduced in RKO cells, the cells were split in 2 populations, 1 population was transduced with Cas13d and the other serving as control did not. The population expressing Cas13d was FACS sorted in low mCherry (efficient gRNAs) and high mCherry (un-efficient gRNAs) in 2 biological replicates and the genomic DNA of these populations was extracted, gRNAs were PCR amplified and sequenced. For the population that did not express Cas13d, a low mCherry, one high mCherry and unsorted population were sequenced as control libraries.
2023-10-29 | E-MTAB-11111 | biostudies-arrayexpress
Project description:Circular RNAs (circRNAs) are widely expressed, but their functions remain largely unknown. To study circRNAs in a high-throughput manner, short hairpin RNA (shRNA) screens1 have recently been used to deplete circRNAs by targeting their unique back-splicing junction (BSJ) sites. Here, we report frequent discrepancies between shRNA-mediated circRNA knockdown efficiency and the corresponding biological effect, raising pressing concerns about the robustness of shRNA screening for circRNA functional characterization. To address this issue, we leveraged the CRISPR/Cas13d system2 for functional study of circRNAs by optimizing the strategy for designing single guide RNAs to deplete circRNAs. We then performed shRNA and CRISPR/Cas13d parallel screenings and demonstrated that shRNA-mediated circRNA screening yielded a high rate of false positive phenotypes. Furthermore, using a CRISPR/Cas13d screening library targeting over 2,500 human hepatocellular carcinomas (HCC) related circRNAs, we identified a group of circRNAs, whose inhibition increased the therapeutic efficacy of sorafenib, an approved HCC drug. Collectively, these data demonstrate that CRISPR/Cas13d system is an effective approach to study the function of circRNAs in a high-throughput manner.
2020-12-06 | GSE162720 | GEO
Project description:Optimized design of antisense oligomers for targeted rRNA depletion
| PRJNA640808 | ENA
Project description:Optimized RNA-targeting CRISPR/Cas13d technology outperforms shRNA in identifying functional circRNAs
| PRJNA682745 | ENA
Project description:Cas13 is a unique family of CRISPR endonucleases exhibiting programmable binding and cleavage of RNAs and is a strong candidate for eukaryotic RNA knockdown in the laboratory and the clinic. However, sequence-specific binding of Cas13 to the target RNA unleashes non-specific bystander RNA cleavage, or collateral activity, which may confound knockdown experiments and raises concerns for therapeutic applications. Although conserved across orthologs and robust in cell-free and bacterial environments, the extent of collateral activity in mammalian cells remains disputed. Here, we investigate Cas13d collateral activity in the context of an RNA-targeting therapy for myotonic dystrophy type 1, a disease caused by a transcribed long CTG repeat expansion. We find that when targeting CUGn RNA in HeLa and other cell lines, Cas13d depletes endogenous and transgenic RNAs, interferes with critical cellular processes, and activates stress response and apoptosis pathways. We also observe collateral effects when targeting other repetitive and unique transgenic sequences, and we provide evidence for collateral activity when targeting highly expressed endogenous transcripts. To minimize collateral activity for repeat-targeting Cas13d therapeutics, we introduce gRNA excision for negative-autoregulatory optimization (GENO), a simple strategy that leverages crRNA processing to control Cas13d expression and is easily integrated into an AAV gene therapy. We argue that thorough assessment of collateral activity is necessary when applying Cas13d in mammalian cells and that implementation of GENO illustrates the advantages of compact and universally robust regulatory systems for Cas-based gene therapies.
2021-12-23 | GSE191329 | GEO
Project description:We explored whether the targeting of crRNAs affected the host cell gene expression. To do this, we used a type LNP, termed a lipitoid, to transiently transfect human A549 lung carcinoma cells with the Cas13d mRNA and in vitro synthesized crRNA(s). At 48 hpi, we isolated cellular RNA and performed whole-cell RNA sequencing (RNA-seq). We observed high reproducibility between biological replicates. We examined the off-target effects of viral-targeting crRNA for SARS-CoV-2 or 229E as well as the NT crRNA by comparing their transcriptome profiles to that of cells expressing Cas13d alone, and we saw highly similar gene expression profiles. Furthermore, co-delivery of Cas13d and two targeting crRNAs showed a very similar transcriptome profile to that of the Cas13d alone. These data suggest that Cas13d and antiviral crRNAs are highly specific, with a minimal impact on host cell transcriptomes.
2022-04-27 | GSE186020 | GEO
Project description:In this work we analyzed crRNA processing by SuCasΩ Type V CRISPR-Cas nuclease in E. coli
2023-01-11 | GSE178531 | GEO