Developing and testing a method to reduce circAdpgk-0001 RNA using CRISPR-Cas13d technology

The JS-1 cell line, a mouse-derived hepatic stellate cell (HSC), was purchased from the Otwo Biotech, China. The cell line was routinely cultured in Dulbecco's Modified Eagle's Medium (DMEM) (Gibco, Beijing, China) supplemented with 10% FBS (ExCell, Shanghai, China) at 37 °C with 5% CO. 2

The mature sequence was obtained according to circAdpgk-0001 (circAtlas, chr9:59297526—59303551). And the BSJ sites of circAdpgk-0001 were analyzed by Circprimer2.0. Then, 11, 12, 14, and 16 nucleotides were taken respectively on both sides centered on BSJ (BSJ was not included), resulting in crRNAs with lengths of 24, 26, 30, and 34 nucleotides. Finally, based on the principle of base complementary pairing, SnapGene was used to design forward complementary single oligonucleotide chain and reverse one for crRNAs with different lengths for subsequent use.

Two µg CRISPR-RfxCas13d plasmid (Addgene, Watertown, MA, USA), two µL BsmBI-v2 enzyme (NEB, USA), five µL 10 × NEBuffer3.1 and water were mixed in a tube to form a 50 µL enzyme digestion system and digested at 55 °C for 1 h. The enzyme digestion products were purified using PCR product purification kit (UEBio, Shanghai, China), and the linearized CRISPR-RfxCas13d vector was obtained. Nine µL of 100 µM forward single oligonucleotide chain, nine µL of 100 µM reverse single oligonucleotide chain and two µL of NEBuffer 2.1 were mixed in a tube. Annealing products were obtained by placing them in a PCR instrument and gradually decreasing from 95 °C to 22 °C at a rate of 1 °C per minute. Three µL annealing product, 50 ng linearized CRISPR-RfxCas13d plasmid, one µL T4 DNA Ligase (Takara, Japan), and one µL 10 × Ligation buffer were mixed in a tube, which was supplemented to 10 µL with water and incubated at 16 °C for overnight. After transformation, individualDH5α colony in the Petri dishes (Ampicillin) was picked up and moved to a tube containing five ml LB. Place the tube in a shaking incubator at 180 rpm for 6–8 h at 37 °C. The bacterial solution was added to 200 ML LB in a shaking incubator at 180 rpm for 14–16 h at 37 °C for expanded culture. Plasmids were extracted using the E.Z.N.A.Endo-free Plasmid Maxi Kit (OMEGA Biotek, , Norcross, GA, USA). Then the plasmid was further confirmed through sequencing. siRNAs of circAdpgk-0001 were synthesized to target the junction site of circAdpgk-0001. Three siRNAs were designed and synthesized by Genepharma and another three siRNAs were designed and synthesized by GeneseedBio. In the experiment, NC-siRNA was used as the control group. All sequences of siRNA used in this study were listed in. E. coli ® Table S1

JS-1 cells were transfected using Lipofectamine3000 (Thermo Fisher Scientific, Waltham, MA, USA) or jetPRIME Transfection (Polyplus, Illkirch, France) according to the manufacturer's protocol. Different transfection conditions were designed to improve the knockdown efficiency. Condition 1: after cells were seeded and reached 70–90% confluent, they were stimulated with TGF-β (10 ng/ml) (Novoprotein, Suzhou, China) for 24 h, Lipofectamine3000 was used for transfection for 24 h. Condition 2: after cells were seeded and reached 70–90% confluent, they were stimulated with TGF-β (10 ng/ml) for 24 h, Lipofectamine3000 was used for transfection for 48 h. Condition 3: after cells were seeded and reached 70–90% confluent, they were stimulated with TGF-β (10 ng/ml) for 24 h, jetPRIME Transfection was used for transfection for 24 h. Condition 4: after cells were seeded and reached 70–90% confluent, Lipofectamine3000 was used for transfection and TGF-β was added at the same time for 24 h. Knockdown efficiency of circAdpgk-0001 was confirmed using qRT-PCR. ® ® ® ®

Total RNA extraction from cells was performed with TRIzol reagent (TransGen, Beijing, China). The RNA concentration was measured using a NanoDrop 2000 (Thermo Fisher Scientific, USA). The entire process was carried out under conditions free from RNase contamination. Complementary DNA was synthesized using Evo M-MLV Reverse Transcriptase kit AG11706 (AGbio, Beijing, China). SYBR Green Premix Pro Taq HS qPCR kit AG11701 (AGbio) and CFX96 RT-PCR system (Bio-Rad, USA) were used for qRT-PCR detection. The two-step method was adopted, which included pre-denaturation at 95 °C for 30 s, followed by 40 cycles of denaturation at 95 °C for 5 s and annealing/extension at 60 °C for 60 s. Three replicate wells were set for each sample. GAPDH was used to normalize the expression of mRNAs. The relative abundance of expression was calculated using the 2method. The target primer sequences were designed using Premier 6. The sequences of primers used in qRT-PCR were listed in. − △△ Ct Table S2

Cells were lysed on ice using RIPA buffer (Beyotime, Haimen, China) supplemented with proteinase inhibitor (Beyotime). Total proteins were extracted and measured using a BCA protein assay kit (Beyotime). Equal amount of protein was subjected to SDS-PAGE electrophoresis separation, and transferred onto PVDF membranes, which were blocked in non-fat milk. Then the membranes were cultured with the primary antibody at 4 °C overnight. Afterwards, the secondary antibody was incubated for 1 h at room temperature. The ECL Substrate Kit (Thermo Fisher Scientific, USA) was applied to visualize the target protein bands. Antibodies dilution was as follows: rabbit monoclonal antibody GAPDH (1:3000; Boster), rabbit monoclonal antibody α-SMA (1:5000, Abcam, USA), rabbit polyclonal antibody Collagen I (1:1000, Boster), and HRP-conjugated goat anti-rabbit antibody (1:10000; ZSGB, Beijing, China). Followed with quantifying by Image J software (NIH, Bethesda, MD, USA) and normalized to GAPDH, the internal control.

All data analysis was performed using GraphPad Prism v7.01 software (GraphPad, La Jolla, CA, USA). The results were exhibited as the mean ± standard deviation. Independent sample-test was used for comparisons between two experimental groups, and one-way analysis of variance (ANOVA) was applied to analyze the difference among multiple groups. Data are mean ± SEM of at least three independent experiments. The-value < 0.05 was considered significant. t p

To knock down circAdpgk-0001, we designed and synthesized six siRNAs targeting circAdpgk_0001 by two biotechnology companies. After transfecting siRNA-1 through siRNA-6 into the JS-1 cell line, only siRNA-5 showed higher knockdown efficiency, reducing circAdpgk-0001 levels by approximately 30% under transfection condition 1 (), which was insufficient for subsequent experiments. Therefore, to further improve the knockdown efficiency, we designed different experimental conditions by modifying three factors: prolonging the transfection time, changing the transfection reagent, and co-transfection with TGF-β. The knockdown efficiency under transfection condition 2 was approximately 20% (). The knockdown efficiency under transfection condition 3 was approximately 40% (). The knockdown efficiency under transfection condition 4 was approximately 30% (). Overall, the siRNAs were able to knock down circAdpgk-0001 by approximately 40% under the optimal experimental conditions. circ-Cyp4f18_0001 was knocked down by approximately 30%, and circ-Cdkn3_0002 by approximately 20% using siRNAs under the optimal conditions (–). Fig. 1A Fig. 1B Fig. 1C Fig. 1D Figs. S1A S1B

Four crRNAs targeting the BSJ of circAdpgk-0001, with nucleotide lengths of 24, 26, 30, and 34, were designed (). Four recombinant plasmids targeting circAdpgk-0001 were generated, named Cas13d/24, Cas13d/26, Cas13d/30, and Cas13d/34, with the empty CRISPR-RfxCas13d plasmid serving as a control. All plasmids were sequenced to confirm the accuracy of their nucleotide sequences. Under transfection condition 1, the control plasmid did not affect the expression of circAdpgk-0001, while Cas13d/24 achieved the knockdown efficiency of approximately 50% (). Prolonged transfection under condition 2 resulted in the knockdown of circAdpgk-0001 by approximately 50% with Cas13d/24 (). After applying transfection condition 3 and changing the transfection reagent, Cas13d/24 resulted in the knockdown of circAdpgk-0001 by approximately 30% (). Under transfection condition 4, after cells were simultaneously transfected with TGF-β, Cas13d/24 resulted in the knockdown of circAdpgk-0001 by approximately 70% (). Furthermore, it was found that the knockdown of circAdpgk-0001 by Cas13d/24 had no effect on the expression of the maternal gene ADPGK (). These results demonstrated that CRISPR-Cas13d RNA targeting technology could effectively knock down circAdpgk-0001 with high efficiency and specificity. Fig. 2A Fig. 2B Fig. 2C Fig. 2D Fig. 2E Fig. 2F

Cell experiments were conducted using the optimal transfection conditions for siRNA-5 and the Cas13d/24 plasmid to compare the knockdown efficiency simultaneously. The knockdown efficiency of the Cas13d/24 recombinant plasmid was significantly higher than that of siRNA-5 (–). The effect of circAdpgk-0001 knockdown on activation-related factors was further investigated in the JS-1 cell line. qRT-PCR results showed that the expression of activation-related factors, collagen I and α-SMA, decreased by approximately 30% after circAdpgk-0001 knockdown with siRNA-5 (). The expression of collagen I and α-SMA decreased by approximately 40% after circAdpgk-0001 knockdown with Cas13d/24 (). We compared the effects of the two methods on collagen I and α-SMA and found that the effects were more significant after Cas13d/24 knockdown of circAdpgk-0001 (). Furthermore, Western blot results revealed that circAdpgk-0001 knockdown also reduced the protein expression of collagen I and α-SMA (–). Moreover, the effect of the Cas13d/24 recombinant plasmid on the protein level of α-SMA was also higher than that of siRNA-5 (). These results indicated that CRISPR-Cas13 RNA targeting technology could significantly knock down circAdpgk-0001 and reduce the expression of activation-related factors in the JS-1 cell line. Figs. 3A 3B Fig. 3C Fig. 3D Fig. 3E Figs. 4A 4B Fig. 4C