Publisher Correction: Gene knockdown via electroporation of short hairpin RNAs in embryos of the marine hydroid Hydractinia symbiolongicarpus

AbstractPerforming gene function analyses in a broad range of research organisms is crucial for understanding the biological functions of genes and their evolution. Recent studies have shown that short hairpin RNAs (shRNAs) can induce gene-specific knockdowns in two cnidarian species. We have developed a detailed, straightforward, and scalable method to deliver shRNAs into fertilized eggs of the hydrozoan cnidarian Hydractinia symbiolongicarpus via electroporation, yielding gene-targeted knockdowns that can be assessed throughout embryogenesis, larval settlement, and metamorphosis. Our electroporation protocol allows for the transfection of shRNAs into hundreds of fertilized H.symbiolongicarpus eggs simultaneously with minimal embryo death and no long-term harmful consequences on the developing animals. We show RT-qPCR and detailed phenotypic evidence of our method successfully inducing significant knockdowns of an exogenous gene (eGFP) and an endogenous gene (Nanos2). We also provide visual confirmation of successful shRNA transfection inside embryos through electroporation. This is the first time that electroporation as a delivery system has been developed for Hydractinia. Our detailed protocol for electroporation of shRNAs in H. symbiolongicarpus embryos constitutes an important experimental resource for the hydrozoan community while also serving as a successful model for the development of similar methods for interrogating gene function in other marine invertebrates.

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Gene knockdown via electroporation of short hairpin RNAs in embryos of the marine hydroid Hydractinia symbiolongicarpus

Scientific Reports ◽

10.1038/s41598-020-69489-8 ◽

2020 ◽

Vol 10 (1) ◽

Cited By ~ 1

Author(s):

Gonzalo Quiroga-Artigas ◽

Alexandrea Duscher ◽

Katelyn Lundquist ◽

Justin Waletich ◽

Christine E. Schnitzler

Keyword(s):

Gene Function ◽

Marine Invertebrates ◽

Endogenous Gene ◽

Successful Model ◽

Hydractinia Symbiolongicarpus ◽

Endogenous Genes ◽

Short Hairpin ◽

Visual Confirmation ◽

Short Hairpin Rnas

Abstract Analyzing gene function in a broad range of research organisms is crucial for understanding the biological functions of genes and their evolution. Recent studies have shown that short hairpin RNAs (shRNAs) can induce gene-specific knockdowns in two cnidarian species. We have developed a detailed, straightforward, and scalable method to deliver shRNAs into fertilized eggs of the hydrozoan cnidarian Hydractinia symbiolongicarpus via electroporation, yielding effective gene-targeted knockdowns that can last throughout embryogenesis. Our electroporation protocol allows for the transfection of shRNAs into hundreds of fertilized H. symbiolongicarpus eggs simultaneously with minimal embryo death and no long-term harmful consequences on the developing animals. We show RT-qPCR and detailed phenotypic evidence of our method successfully inducing effective knockdowns of an exogenous gene (eGFP) and an endogenous gene (Nanos2), as well as knockdown confirmation by RT-qPCR of two other endogenous genes. We also provide visual confirmation of successful shRNA transfection inside embryos through electroporation. Our detailed protocol for electroporation of shRNAs in H. symbiolongicarpus embryos constitutes an important experimental resource for the hydrozoan community while also serving as a successful model for the development of similar methods for interrogating gene function in other marine invertebrates.

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Large-Scale, High-Throughput Validation of Short Hairpin RNA Sequences for RNA Interference

CrossRef Listing of Deleted DOIs ◽

10.1177/1087057105284342 ◽

2006 ◽

Vol 11 (3) ◽

pp. 236-246 ◽

Cited By ~ 6

Author(s):

Laurence H. Lamarcq ◽

Bradley J. Scherer ◽

Michael L. Phelan ◽

Nikolai N. Kalnine ◽

Yen H. Nguyen ◽

...

Keyword(s):

High Throughput ◽

Large Scale ◽

Strong Support ◽

Gc Content ◽

Rapid Identification ◽

Hairpin Rna ◽

Rna Sequences ◽

Short Hairpin ◽

Short Hairpin Rnas ◽

Interfering Rna

A method for high-throughput cloning and analysis of short hairpin RNAs (shRNAs) is described. Using this approach, 464 shRNAs against 116 different genes were screened for knockdown efficacy, enabling rapid identification of effective shRNAs against 74 genes. Statistical analysis of the effects of various criteria on the activity of the shRNAs confirmed that some of the rules thought to govern small interfering RNA (siRNA) activity also apply to shRNAs. These include moderate GC content, absence of internal hairpins, and asymmetric thermal stability. However, the authors did not find strong support for positionspecific rules. In addition, analysis of the data suggests that not all genes are equally susceptible to RNAinterference (RNAi).

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Optimized RNA-targeting CRISPR/Cas13d technology outperforms shRNA in identifying functional circRNAs

Genome Biology ◽

10.1186/s13059-021-02263-9 ◽

2021 ◽

Vol 22 (1) ◽

Cited By ~ 1

Author(s):

Yang Zhang ◽

Tuan M. Nguyen ◽

Xiao-Ou Zhang ◽

Limei Wang ◽

Tin Phan ◽

...

Keyword(s):

Hepatocellular Carcinoma ◽

High Throughput ◽

Biological Effect ◽

Human Hepatocellular Carcinoma ◽

Guide Rnas ◽

Rna Targeting ◽

Short Hairpin ◽

Short Hairpin Rnas ◽

Screening Library ◽

High Throughput Study

AbstractShort hairpin RNAs (shRNAs) are used to deplete circRNAs by targeting back-splicing junction (BSJ) sites. However, frequent discrepancies exist between shRNA-mediated circRNA knockdown and the corresponding biological effect, querying their robustness. By leveraging CRISPR/Cas13d tool and optimizing the strategy for designing single-guide RNAs against circRNA BSJ sites, we markedly enhance specificity of circRNA silencing. This specificity is validated in parallel screenings by shRNA and CRISPR/Cas13d libraries. Using a CRISPR/Cas13d screening library targeting > 2500 human hepatocellular carcinoma-related circRNAs, we subsequently identify a subset of sorafenib-resistant circRNAs. Thus, CRISPR/Cas13d represents an effective approach for high-throughput study of functional circRNAs.

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