Establishment and evaluation of a circAdpgk-0001 knockdown method using CRISPR-Cas13d RNA-targeting technology

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.