Related Experiment Video
Updated: Sep 13, 2025
Rearing and Double-stranded RNA-mediated Gene Knockdown in the Hide Beetle, Dermestes maculatus
Published on: December 28, 2016
Cas13d-mediated isoform-specific RNA knockdown with a unified computational and experimental toolbox
Megan D Schertzer1,2, Andrew Stirn3,4, Keren Isaev3,5
1New York Genome Center, New York, NY, USA. megan.schertzer@gmail.com.
Abstract:
Pre- and post-transcriptional mechanisms, including alternative promoters, termination signals, and splicing, play essential roles in diversifying protein output by generating distinct RNA and protein isoforms. Two major challenges in characterizing the cellular function of alternative isoforms are the lack of experimental methods to specifically and efficiently modulate isoform expression and computational tools for complex experimental design and analysis. To address these gaps, we develop and methodically test an isoform-specific knockdown strategy which pairs the RNA-targeting CRISPR/Cas13d system with guide RNAs that span exon-exon junctions. In parallel, we provide computational tools for experimental design and analysis. In this study, we demonstrate that junction-targeting achieves robust and isoform-specific RNA knockdown across diverse alternative isoform events, genes, and cell types.
Insights
We developed a new method using CRISPR/Cas13d to specifically reduce RNA and protein isoforms. This technique enables precise control over gene expression for studying cellular functions.
Area of Science:
- Molecular Biology
- Genetics
- Biotechnology
Background:
- Alternative splicing and other mechanisms generate diverse RNA and protein isoforms, crucial for cellular functions.
- Characterizing isoform function is challenging due to limited methods for specific expression modulation and complex data analysis.
Purpose of the Study:
- To develop and validate an efficient, isoform-specific RNA knockdown strategy.
- To provide computational tools supporting experimental design and analysis for isoform studies.
Main Methods:
- Developed an RNA-targeting CRISPR/Cas13d system utilizing guide RNAs spanning exon-exon junctions.
- Tested the strategy across various genes, alternative splicing events, and cell types.
- Created accompanying computational tools for experimental design and data analysis.
Main Results:
- Demonstrated robust and isoform-specific RNA knockdown using the junction-targeting CRISPR/Cas13d system.
- Validated the method's effectiveness across diverse alternative splicing events and cell lines.
- Showcased the utility of the developed computational tools for experimental workflows.
Conclusions:
- The developed junction-targeting CRISPR/Cas13d system provides a powerful tool for isoform-specific gene knockdown.
- This method overcomes previous limitations in studying alternative isoform function.
- Enables precise investigation of the roles of distinct RNA and protein isoforms in cellular processes.
Related Concept Videos
02:15Experimental RNAi
01:23RNA Interference
This process occurs naturally in cells, often through the activity of genomically-encoded microRNAs. Researchers can take advantage of this mechanism by introducing synthetic RNAs to deactivate specific genes for research or therapeutic purposes. For example, RNAi could be used...
02:30siRNA - Small Interfering RNAs
In the cytoplasm, siRNA is processed from a double-stranded RNA, which comes from either endogenous DNA transcription or exogenous sources like a virus. This double-stranded RNA is then cleaved by the...
02:30Small interfering RNAs (siRNA)