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Establishment and evaluation of a circAdpgk-0001 knockdown method using CRISPR-Cas13d RNA-targeting technology
Sijia Huang1, Hailan Qin1, Bingxin Dai1,2
1Department of Microbiology and Parasitology, Anhui Province key Laboratory of Zoonoses, School of Basic Medical Sciences, Anhui Medical University, Hefei, China.
Background:
The small interfering RNA (siRNA) method has been used to knock down circular RNAs (circRNAs). However, issues such as low efficiency and off-target effects have become increasingly recognized. Recent studies have demonstrated that CRISPR-Cas13 can specifically target and cleave RNA. In this study, we established a CRISPR-Cas13d-based RNA-targeting method to specifically knock down circRNAs, such as circAdpgk-0001, and compared its performance with the siRNA method.
Methods:
Four clustered regularly interspaced short palindromic repeats (CRISPR) RNAs (crRNAs) of different nucleotide lengths spanning the back-splicing junction (BSJ) of circAdpgk-0001 were designed. A CRISPR-RfxCas13d plasmid capable of specifically cleaving circAdpgk-0001 was constructed and transfected into the JS-1 cell line. Knockdown efficiency was assessed using quantitative real-time PCR (qRT-PCR) and compared with that of the siRNA method. The expression of activation-related factors alpha-smooth muscle actin (α-SMA) and collagen I in JS-1 cells was further evaluated using qRT-PCR and Western blot.
Results:
CRISPR-Cas13d with a 24-nucleotide crRNA showed the highest knockdown efficiency (∼50%). After further optimization, the knockdown efficiency of CRISPR-Cas13d reached 70%, significantly higher than that of the siRNA method (40%). Knockdown of circAdpgk-0001 using Cas13d reduced the expression of collagen I and α-SMA by approximately 40%, which was greater than the reduction achieved by siRNA-mediated knockdown.
Conclusion:
CRISPR-Cas13d demonstrated higher efficiency than the siRNA method in knocking down circRNAs, providing a promising tool for investigating circRNA functions.
Insights
CRISPR-Cas13d effectively knocks down circular RNAs (circRNAs) with higher efficiency than small interfering RNA (siRNA). This advanced RNA-targeting method offers a promising tool for studying circRNA functions.
Area of Science:
- Molecular Biology
- Genetics
- Biotechnology
Background:
- Small interfering RNA (siRNA) is used for circular RNA (circRNA) knockdown but suffers from low efficiency and off-target effects.
- CRISPR-Cas13 systems offer specific RNA targeting and cleavage capabilities.
- CRISPR-Cas13d presents a novel approach for targeted circRNA knockdown.
Purpose of the Study:
- To establish and evaluate a CRISPR-Cas13d-based method for specific circRNA knockdown.
- To compare the efficiency of CRISPR-Cas13d with the traditional siRNA method for circRNA knockdown.
- To investigate the impact of circRNA knockdown on the expression of collagen I and alpha-smooth muscle actin (α-SMA).
Main Methods:
- Designed clustered regularly interspaced short palindromic repeats (CRISPR) RNAs (crRNAs) targeting the back-splicing junction of circAdpgk-0001.
- Constructed a CRISPR-RfxCas13d plasmid for circAdpgk-0001 cleavage and transfected it into JS-1 cells.
- Assessed knockdown efficiency using quantitative real-time PCR (qRT-PCR) and Western blot, comparing CRISPR-Cas13d with siRNA.
Main Results:
- CRISPR-Cas13d with a 24-nucleotide crRNA achieved ~50% knockdown efficiency, optimized to 70%.
- The siRNA method achieved 40% knockdown efficiency, significantly lower than CRISPR-Cas13d.
- Knockdown of circAdpgk-0001 using CRISPR-Cas13d reduced collagen I and α-SMA expression by ~40%, exceeding siRNA's effect.
Conclusions:
- CRISPR-Cas13d demonstrates superior efficiency compared to siRNA for circRNA knockdown.
- This CRISPR-Cas13d-based method is a potent tool for functional studies of circRNAs.
- The findings highlight the potential of CRISPR-Cas13d in molecular biology research and therapeutic applications.
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