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shRNA/gRNA Construction
Our shRNA/gRNA plasmid construction services cover RNAi-mediated gene knockdown and CRISPR/Cas9 gene editing, with support for multiple promoters, selectable markers, and reporter combinations.
Services include target efficiency scoring, genome-wide off-target analysis, Sanger sequencing verification, and a complete design report.
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  • 5-10 days

    Standard turnaround time

  • 200+

    In-stock vectors

  • Sanger

    Sequencing verification of the insert region

  • 3-5targets

    Recommended top-scoring candidate targets

首页 > Products > Plasmid Construction > shRNA/gRNA Construction >
Introduction
Product Comparison
Target Sequence Design and Scoring System
Service Advantages
Service Details
Optional Add-On Services
Service Workflow
Delivery Standards
Quality Control Standards
Applications
FAQ
shRNA Plasmid Construction
gRNA Plasmid Construction

shRNA Plasmid Construction Service

Efficient Knockdown, Precise Silencing

Short hairpin RNA (shRNA) is a small RNA molecule expressed from a vector. In cells, it is processed by Dicer into mature siRNA and then assembled into the RISC complex to specifically recognize and degrade target mRNA at the post-transcriptional level, thereby achieving gene knockdown.


We provide shRNA vector systems with multiple promoter options (U6, H1, CMV, etc.), selection markers (puromycin, hygromycin, etc.), and reporter genes (GFP, mCherry, Luciferase, etc.). Customers may provide a Gene ID or Accession Number, and we will complete the design, construction, sequencing verification, and report delivery.

gRNA Plasmid Construction Service

Precise Guidance, Efficient Editing

Guide RNA (gRNA/sgRNA) is the core targeting component of the CRISPR/Cas9 system. After forming a complex with Cas9, it introduces a DNA double-strand break at a specific genomic site, enabling gene knockout, knock-in, or point mutation through NHEJ or HDR.


We support flexible combinations of U6, T7, and other promoters with selection markers and fluorescent reporter genes for mammalian cells, plants, and microbial systems. Our proprietary algorithm evaluates cleavage efficiency, off-target risk, and site specificity to recommend 3–5 highly ranked candidate gRNA target sites and provide an off-target analysis report.

shRNA vs. gRNA: How to Choose

Compare the level of action, mechanism, durability, and applications
Comparison shRNA (RNAi) gRNA/sgRNA (CRISPR Editing)
Level of Action Post-transcriptional (mRNA degradation) DNA level (genome cleavage)
Mechanism shRNA → Dicer processing → siRNA → RISC-mediated mRNA degradation gRNA guides Cas9 to cleave double-stranded DNA → NHEJ/HDR repair
Type of Effect Gene knockdown (partial suppression) Gene knockout/knock-in/editing
Durability Vector-expression dependent and reversible (recovers after selection is removed) Permanent DNA alteration that can be inherited by daughter cells
Target Sequence Length 19–29 bp (targets mRNA) Approximately 20 bp + PAM (NGG, targets genomic DNA)
Cas9 Requirement Not required Required (co-transfection with Cas9 plasmid/protein/mRNA)
Off-Target Risk Seed-sequence homology-related off-target effects (generally milder) Guide-sequence mismatch-related off-target effects (requires rigorous evaluation)
Applications Gene function studies, signaling pathways, reversible silencing, and library screening Gene knockout, disease models, gene therapy, and point-mutation correction
Vector Promoter Primarily Pol III promoters (U6/H1) U6 promoter (gRNA) + Pol II promoter (Cas9)

Selection Recommendation:Choose shRNA for reversible gene silencing, essential-gene studies, or high-throughput screening. Choose gRNA for permanent gene knockout, disease modeling, gene therapy, gene knock-in, or precise point mutation (precise editing requires an HDR template).

Target Sequence Design and Scoring System

Multi-parameter efficiency scoring, genome-wide alignment, and rigorous off-target analysis

shRNA Target Sequence Design:The sense-strand seed region (positions 2–8) is aligned against non-target mRNA 3′ UTRs across the transcriptome. Combined with miRNA-like off-target assessment and BLAST genome-wide alignment, highly homologous sequences are excluded. Three independent target sequences are designed for each gene (three-to-one strategy), with qPCR verification of at least one sequence achieving ≥50% knockdown.

gRNA Target Sequence Design:Cleavage efficiency is evaluated using algorithms such as Doench Rule Set 2/Azimuth. Mismatch tolerance in the PAM-proximal region is systematically analyzed, while conserved exons are prioritized and common SNP sites are avoided. Two to three candidate gRNAs are designed for each gene together with validation primers.

Design and Scoring Workflow:Target gene information → Candidate sequence generation → Specificity and efficiency evaluation → Genome-wide off-target analysis → Secondary structure assessment → Selection of 2–3 candidate sequences with a complete scoring and off-target report.

Service Advantages

Rapid construction, rigorous scoring, sequencing verification, and broad species compatibility
  • Rapid Delivery · Professional Team
    A dedicated team and mature direct-ligation platform support delivery of standard projects in 5–10 days, with competitive pricing and a short turnaround time.
  • Rigorous Scoring · High-Efficiency Target Sequences
    shRNA designs undergo specificity assessment and off-target analysis using a three-to-one strategy: at least one sequence achieves ≥50% knockdown. gRNA designs use Doench Rule Set 2 and genome-wide off-target analysis.
  • Insert-Region Sequencing Verification
    The target-sequence insertion region is verified by sequencing, with sequencing chromatograms and alignment files provided.
  • 200+ In-Stock Vectors · Ready to Select
    Options include U6, H1, CMV, T7, and other promoters; puromycin, hygromycin, and other selection markers; and GFP, mCherry, Luciferase, and other reporter genes.
  • Broad Species Support · Flexible Compatibility
    gRNA design supports humans, mice, rats, zebrafish, plants, and other species; shRNA design supports common species such as humans, mice, and rats.

Service Details

Custom shRNA/gRNA, three-to-one design, and dual-target construction
Service Type Target Sequence Length Turnaround Time
(calendar days)		 Price Notes
Custom shRNA/gRNA
 19-25 bp 5-10
Contact Us
 Customer-provided target sequence
or one sequence designed by us
gRNA Three-to-One
 19-25 bp 5-12
Contact Us
 Three sequences designed per gene,
for candidate screening
shRNA Three-to-One
 19-25 bp 5-12
Contact Us
 At least one sequence
with knockdown efficiency ≥50%
Dual-Target gRNA Construction
 19-25 bp ×2 7-14
Contact Us
 Two gRNAs constructed in one vector,
for large-fragment deletion

Optional Add-On Services

Target sequence design and multiple plasmid preparation options
Add-On ServiceNotesTurnaround
Target Sequence DesignReynolds scoring (shRNA)/Doench scoring (gRNA) + off-target analysis1–2 business days
Endotoxin-Free Plasmid PreparationSuitable for transfection-grade plasmid requirements; endotoxin<0.1 EU/μg3–5 business days
Large-Scale Plasmid PreparationMidi(>100μg) / Maxi(>500μg)5–7 business days

Service Workflow

Requirement submission, proposal and quotation, experimental execution, delivery, and after-sales support
  • Submit Requirements
    Provide the Gene ID or Accession Number and specify the species, vector type, promoter, selection marker, and reporter gene requirements.
  • Proposal and Quotation
    Target design results, vector proposal, off-target prediction, turnaround time, and quotation are provided within 1 business day.
  • Experimental Execution
    Complete target-sequence synthesis, vector construction, transformation screening, and sequencing verification, with traceable progress throughout.
  • Delivery and After-Sales Support
    Deliver recombinant plasmids and glycerol stocks together with complete experimental and design reports, plus technical support for subsequent viral packaging and related services.

Delivery Standards

Complete delivery of plasmids, strains, sequences, sequencing data, maps, and quality reports
  • Plasmid DNA
    Deliver 5 μg of high-purity plasmid DNA containing the target insert, available as lyophilized powder or liquid.
  • Glycerol Stock (Optional)
    Provide E. coli DH5α or a specified host glycerol stock carrying the correct plasmid, stored at −80°C.
  • Standard Sequence Files
    Provide the final plasmid sequence in FASTA/GenBank format with complete annotation of the insert and key vector elements.
  • Sequencing Results
    Provide raw Sanger sequencing chromatograms (.ab1) and a complete sequence alignment report.
  • Plasmid Map
    Provide a complete map annotated with the origin of replication, resistance gene, promoter, multiple cloning site, and insert.
  • COA Quality Report
    Record key parameters including plasmid concentration, purity (A260/A280 ratio), and delivery quantity.

Quality Control Standards

Multi-level quality control from design through plasmid delivery
QC StepTest MethodAcceptance Criteria
Target-Sequence Insert AccuracySanger sequencing (bidirectional sequencing across the insertion site)Insertion region verified by sequencing
Vector IntegrityRestriction digest verification/colony PCRFragment size matches the expected result
Plasmid PurityUV spectrophotometryA260/A280 = 1.8 ~ 2.0
Plasmid ConcentrationUV absorbance≥5 μg (standard delivery amount)
Strain ViabilityGlycerol-stock recovery cultureNormal colony growth after recovery
Off-Target Analysis (Design Stage)Genome-wide alignmentHigh-risk off-target sites excluded
Endotoxin LevelLAL assay (endotoxin-free specification)< 0.1 EU/μg

Applications

For functional studies, disease models, screening, gene therapy, and breeding

Gene Function Studies (shRNA/gRNA)

shRNA is suitable for reversible knockdown studies, while gRNA is suitable for permanent loss-of-function studies involving cell proliferation, apoptosis, differentiation, and migration.

Signaling Pathway and Regulatory Network Analysis

Knock down key pathway nodes or use dual gRNAs to delete gene clusters or regulatory regions, enabling analysis of pathway crosstalk and compensatory mechanisms.

Disease Model Construction

Use CRISPR-Cas9/gRNA to introduce disease-associated mutations and combine with an HDR template for precise point-mutation knock-in.

High-Throughput Screening

shRNA libraries are suitable for negative screening, while gRNA libraries provide broader coverage and support both positive and negative functional genomic screening.

Gene Therapy

Deliver shRNA via AAV and other viral vectors to silence disease-causing genes, or deliver gRNA to correct pathogenic mutations.

Conditional Gene Regulation

Tet-On/Tet-Off inducible shRNA enables temporally controlled knockdown: Dox initiates knockdown, and expression recovers after Dox withdrawal.

Synthetic Biology and Breeding

Applied to metabolic pathway reconstruction, functional validation of engineered strains, crop trait improvement, and breeding of stress-resistant varieties.

FAQ

Common questions about tool selection, target design, vectors, validation, and experimental timelines
What is the difference between shRNA and gRNA, and which should I choose?
shRNA uses RNAi to achieve reversible gene knockdown at the mRNA level, typically with 70–90% suppression. gRNA guides Cas9 to achieve permanent knockout, knock-in, or editing at the DNA level. Choose shRNA for reversible silencing or essential-gene studies, and gRNA for permanent knockout or precise editing.
What does the shRNA “three-to-one” strategy mean, and can it ensure efficacy?
Three independent shRNAs are designed and constructed for one gene, with at least one achieving ≥50% knockdown. The success rate of a single shRNA is approximately 60–70%, while the three-sequence combination can achieve a success rate of over 95%. If none of the three sequences covered by the efficacy assurance meets the standard, they will be redesigned at no additional charge.
How are gRNA targets designed and off-target risks controlled?
Algorithms such as Doench Rule Set 2 are used to predict efficiency. SpCas9 targets are confirmed to have a 3′ NGG PAM, high-risk genomic sites with up to three mismatches are excluded, constitutive exons are prioritized, and SNPs are avoided. A complete off-target analysis report is provided.
Which vectors are available, and how should promoters and selection markers be chosen?
For shRNA, U6/H1 or CMV/EF1a promoters are available; selection markers include Puromycin, Hygromycin, Neomycin, and Blasticidin; reporter genes include eGFP and mCherry. gRNA vectors are available in gRNA-only and all-in-one formats.
Can you construct the insert in my own vector?
Yes. Customers should provide the vector map, sequence, and sequencing verification results, or provide the physical vector for sequencing by us. We will evaluate restriction-site compatibility and promoter suitability and provide recommendations.
How should the result be validated after construction?
For shRNA, mRNA can be measured by qPCR 48–72 hours after transfection, and protein can be measured by Western blot after 72–96 hours. For gRNA, T7E1 digestion or Sanger sequencing plus ICE analysis can be used, together with Western blot to verify protein loss.
What is the difference between shRNA and siRNA?
siRNA is a chemically synthesized double-stranded RNA that acts for 3–7 days and is suitable for short-term experiments. shRNA is continuously expressed from a DNA vector and can be used to generate stable cell lines, package viruses, and support long-term studies and library screening.
How soon can knockout effects be observed after gRNA construction?
DNA cleavage occurs 24–48 hours after gRNA + Cas9 co-transfection, and editing efficiency can be measured after 48–72 hours. Protein levels typically begin to decrease after 72–96 hours. Screening for homozygous monoclonal cells takes approximately 2–4 weeks.

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