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

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.

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.

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.

CRISPR-Cas13d demonstrated higher efficiency than the siRNA method in knocking down circRNAs, providing a promising tool for investigating circRNA functions.