shRNA uses RNA interference to degrade the target transcript at the mRNA level, producing partial, reversible gene knockdown. gRNA guides Cas9 to cleave genomic DNA, enabling complete, permanent, and heritable knockout or knock-in. Choose shRNA when reversible silencing is needed or when studying an essential gene. Choose gRNA for permanent knockout, disease-model construction, or precise editing. If you are unsure, shRNA can be used for initial screening before confirmation with gRNA.

siRNA is a chemically synthesized double-stranded RNA that acts after direct transfection. It has a rapid onset, usually within 24–72 hours, but the effect is transient and typically lasts 3–7 days. shRNA is a hairpin RNA expressed from a DNA vector and processed by Dicer into siRNA. It can be stably expressed from a plasmid or integrated by a viral vector for long-term silencing. Choose siRNA for short-term knockdown and shRNA for long-term studies or stable cell-line generation.

Three independent sequences are designed for one target gene and constructed as three plasmids, with at least one guaranteed to meet the specified performance target: shRNA knockdown efficiency ≥50% or siRNA mRNA inhibition ≥70%. The success rate of an individual sequence is approximately 60–70%, while the combined success rate of three sequences can exceed 95%. If the specified target is not achieved and quantitative data are provided, the sequences will be redesigned at no additional charge.

Algorithms such as Doench Rule Set 2 and Azimuth are used to predict cleavage efficiency. The 3′-NGG PAM for SpCas9 is identified, and genome-wide off-target comparison is performed to exclude high-risk sites. Constitutive exons are prioritized, high-frequency SNP regions are avoided, and secondary structure is evaluated. A complete off-target analysis report is provided. High-fidelity variants such as eSpCas9 or HiFi Cas9 are also recommended to further reduce off-target effects.

shRNA vectors can use U6/H1 (Pol III) or CMV/EF1α (Pol II) promoters. Selection markers include puromycin, hygromycin, neomycin, and blasticidin, while reporter genes include eGFP, mCherry, and luciferase. gRNA vectors are available as gRNA-only formats, which require a separate Cas9 source, or all-in-one formats that co-express gRNA and Cas9. For transient transfection, choose a non-viral vector with puromycin and eGFP; for stable cell-line generation, choose a lentiviral vector with puromycin; and for one-stop editing, choose an all-in-one vector.

For shRNA, measure the reduction in mRNA by qPCR 48–72 hours after transfection and the reduction in protein by Western blot 72–96 hours after transfection. For gRNA, extract genomic DNA 48–72 hours after transfection and assess editing rapidly by a T7E1 assay, or use Sanger sequencing with ICE analysis to determine the indel frequency. Protein loss can also be confirmed by Western blot. If a homozygous knockout clone is required, single-cell cloning by limiting dilution can be performed, which usually takes about 2–4 weeks.

Yes. Please provide the vector map, complete sequence, and sequencing verification result, or send the physical plasmid for sequencing. We will assess restriction-site compatibility and confirm whether the promoter is suitable. If your vector contains only a Pol II promoter, an additional Pol III promoter must be introduced to drive shRNA expression.

DNA cleavage occurs 24–48 hours after co-transfection, and editing efficiency can be assessed by T7E1 digestion or sequencing after 48–72 hours. With an all-in-one vector, there is no need to wait for a separate Cas9 source, and detection can begin at 48 hours. At the protein level, additional time is required for pre-existing protein to degrade; a reduction is generally visible by Western blot after 72–96 hours.