Cut. Through. Limits.
OmniGuide RNA

OmniGuide Design & Order Center

Online design currently available for Cas9 sgRNA, Cas12a crRNA, and pegRNA.

Need help from an expert?

CRISPR Catalog Products

Validate your experimental system and optimize editing efficiency with our off-the-shelf catalog products:

Interested in cGMP synthesis?

We also offer cGMP manufacturing to support your transition from research to clinic. Our state-of-the-art cGMP production facility produces clinical-grade synthetic gRNA for CRISPR/Cas9, CRISPR/Cas12a, prime editing, base editing, and more- with flexible batch sizes (40 mg to grams) and full compliance with FDA, EMA, PMDA, and NMPA guidelines. Comprehensive QA/QC documentation, validated QC testing, and regulatory support, including a US Type II DMF, ensure your materials meet the highest standards for clinical development.

An Rx-360 Audit Report (JA-3136) is also available for licensing—ensuring regulatory confidence without the need for an on-site audit.

Learn More

Case Studies

OmniGuide RNA delivers proven editing efficiency across diverse editing systems

Our HPLC-purified CRISPR/Cas9 sgRNA delivers consistently high editing efficiency, up to 97%, across diverse loci and cell types.

Our Cas12a crRNA enables high knock-out efficiency with multiple Cas12a enzymes, achieving up to 98% editing in Jurkat cells.

Our desalt and HPLC-purified pegRNA achieved up to 52.5% and 63.5% editing, respectively, with efficiencies increasing in a dose-dependent manner.

Our base editing gRNA with CBEmax mRNA achieved 96.8% C-to-T conversion.

Our base editing gRNA with ABE8a protein achieved 97.0% A-to-G conversion.

Resources

A Beginner’s Guide to Guide RNA for Advanced Gene Editing

Learn about the gRNA elements enabling next‑gen editing techniques and more.

Free Download

RNP user manual

A guide on how to use CRISPR RNP for targeted genome editing.

Free Download

CRISPR Lipofection Protocol for HEK293T/293 cells using Lipofectamine™ 2000 and CRISPRMAX™ Cas9 Transfection Reagents

Free Download

Cas12a Editing Protocol for HEK 293T/293 Cells Using the Thermo Fisher Neon® Transfection System

Free Download

Prime Editing Protocol for HEK293T cells with Thermo Fisher Neon® Transfection System

Free Download

Base Editing Protocol for Jurkat cells with Thermo Fisher Scientific Neon™ NxT Electroporation System

Free Download

Webinar: Boost CRISPR Editing Efficiency with Optimized sgRNA & HDR Template Design

Presenter: Dr. Jie Zhu
Field Application Scientist, GenScript

Learn More

OmniGuide — FAQ

• How do I design an effective gRNA for my target gene?

  Designing an effective gRNA depends on several factors, including specificity, efficiency, and compatibility with your CRISPR system. General guidelines include:

  • Choose a sequence complementary to your target DNA and containing the appropriate protospacer adjacent motif (PAM) for your Cas nuclease.
  • Avoid sequences with high similarity to non-target regions to minimize off-target effects. A good gRNA has a high on-target score and low off-target score, which our design tools rank accordingly.
  • Avoid Poly-T sequences (≥4 consecutive Ts) as they terminate transcription
  • Minimize hairpin formation in the gRNA.
  • For Cas9: optimize GC content and spacer length (generally 40–80% GC and 17–24 nucleotides). Cas12a also benefits from moderate GC content, though the optimal range is less strict.
  • Base and prime editing require additional considerations:

    Base editing: the target base must fall within the editing window (~positions 4–8 for cytosine editors, ~4–7 for adenine editors).

    Prime editing: pegRNA include a primer binding site (PBS) and a reverse transcriptase template (RTT), each with specific design constraints (PBS length, melting temperature, etc.).

  To streamline your design process, you can use our OmniGuide RNA Design Tool to generate Cas9 sgRNA, Cas12a crRNA, or pegRNA, or our HDR Knock-In Design Tool for designing Cas9 or Cas12a guides together with a customized HDR template for knock-in experiments.

• How do I choose the best gRNA from the list generated by your design tools?

  The ranking of gRNA designs generated by GenScript's design tools is based on factors that may affect knockout (KO) efficiency, including the comprehensive On-target score, Off-target score, transcript coverage, targeting of early exons, and GC content between 40–80%. We recommend selecting the top three ranked gRNAs directly. If On-target or Off-target scores are of particular concern, you may also weigh these factors more heavily in your selection.

• Does GenScript validate all gRNAs generated by your design tools?

  No, the gRNA sequences generated by our design tools are optimized using computational algorithms to maximize on-target efficiency and minimize off-target effects, but they have not been experimentally validated. Functional testing—such as evaluating the guides in cells or with relevant assays—will still be necessary once you receive your gRNA.

  While not every individual gRNA is experimentally tested, GenScript’s design process is supported by robust algorithmic predictions, extensive validation datasets, and quality-controlled manufacturing. Additionally, we provide tools and resources to help customers perform their own validation experiments.

• How do I validate gRNA efficiency and editing outcomes?

  Here is a comprehensive guide for validating gRNA efficiency and editing outcomes:

  1. Initial Efficiency Assessment

  T7 Endonuclease I (T7E1) Assay: A quick, cost-effective screening method.

  PCR amplify the target region, denature/reanneal, and digest with T7E1. Cleaved products indicate indel formation.

  Provides rough quantification (detection range: ~5–50%).

  Surveyor Assay: Similar principle to T7E1 but more sensitive; better for detecting low-frequency edits.

  TIDE Analysis: Sanger sequencing–based quantification that provides precise indel frequencies and patterns.

  2. High-Resolution Analysis

  Next-Generation Sequencing (NGS): Amplicon sequencing of the target region provides detailed indel spectra and frequencies; can detect rare events (<1%).

  Restriction Fragment Length Polymorphism (RFLP): Applicable if editing disrupts or creates restriction sites; simple gel-based readout but limited to specific sequence changes.

  3. Protein-Level Validation

  Western Blot Analysis: Confirms protein knockout/knockdown; use multiple antibodies whenever possible.

  Flow Cytometry: For surface proteins or fluorescent markers; enables quantitative assessment of editing efficiency at single-cell resolution.

  Immunofluorescence: Assesses cellular localization and expression changes, combining protein detection with morphological analysis.

  4. Functional Validation

  Phenotypic Assays: Include cell viability, proliferation, and migration assays.

  Metabolic Activity Measurements.

  Drug Sensitivity Testing.

  Reporter Systems:

  GFP disruption assays for quick screening

  Traffic light reporters for HDR efficiency

  Split-fluorescent protein complementation assays

  5. Precise Editing Validation (HDR/Base Editing)

  Allele-Specific PCR:
  Design primers specific to the edited sequence.
  Confirms successful integration of desired changes.

  Digital PCR (dPCR):
  Highly quantitative measurement of editing ratios.
  Superior sensitivity for low-frequency events.

  Long-Range PCR:
  Verifies large insertions and ensures surrounding regions remain intact.
  Checks for unintended rearrangements.

  6. Off-Target Assessment

  Predicted Site Analysis: PCR amplify and sequence the top predicted off-targets; use computational tools such as CasOFFinder or CHOPCHOP.

  Unbiased Detection: Employ DISCOVER-Seq, CIRCLE-seq, or GUIDE-seq for genome-wide off-target profiling.

  7. Quality Control Measures

  Multiple Time Points: Assess editing at 24 h, 48 h, 72 h, and 1 week to monitor editing kinetics and stability.

  Clonal Analysis: Isolate single-cell clones for homogeneous populations; allow detailed characterization of individual clones. Essential for generating stable cell lines.

  Multiple gRNAs: Test 2–3 gRNAs per target; consistent results strengthen confidence in on-target specificity and help rule out off-target effects.

  8. Statistical Considerations

  Experimental Design: Include appropriate controls (mock, non-targeting gRNA). Use sufficient biological replicates (n ≥ 3) and technical replicates for assay validation.

  Data Analysis: Apply appropriate statistical tests, correct for multiple comparisons when relevant, and report confidence intervals.

  Recommended Validation Workflow

  Phase 1: Initial Screening (24–48 h post-editing)
  T7E1 or TIDE analysis for efficiency
  Western blot for protein knockdown (if applicable)
  Cell viability assessment

  Phase 2: Detailed Characterization (72 h–1 week)
  NGS amplicon sequencing for precise quantification
  Functional assays relevant to the target gene
  Check top 3–5 predicted off-target sites

  Phase 3: Long-term Validation (1–4 weeks)
  Clonal analysis of edited populations
  Stability assessment over multiple passages
  Comprehensive phenotypic characterization

  Method Selection Guide

  For screening multiple gRNAs: T7E1 or TIDE
  For precise quantification: NGS amplicon sequencing
  For protein validation: Western blot plus functional assays
  For HDR validation: Allele-specific PCR plus Sanger sequencing
  For off-target assessment: Targeted sequencing of predicted sites

• How do I minimize off-target effects in CRISPR editing?

  To minimize off-target effects:

  • Carefully design guides with balanced GC content and minimal similarity to other genomic sites. Consider truncated or chemically modified guides.
  • Use high-fidelity nucleases (e.g., eSpCas9), deliver CRISPR as RNPs for transient activity, and titrate nuclease and guide concentrations.
  • For highly precise edits, base or prime editors may reduce unwanted changes:
    • Base Editors
      

      Mechanism: Base editors employ a modified Cas protein fused to a deaminase enzyme to directly convert one DNA base into another (e.g., C→T or A→G) without generating double-strand breaks (DSBs).

      Reduced Off-Target Effects:
      No DSBs: Traditional CRISPR-Cas9 creates DSBs, which can lead to unintended insertions or deletions (indels) at both on-target and off-target sites. Base editors avoid this, minimizing unwanted mutations.

      Higher Specificity: The editing window is narrow, and the deaminase modifies only specific bases, reducing the likelihood of off-target changes.

    • Prime Editors
      

      Mechanism: Prime editors use a Cas9 nickase fused to a reverse transcriptase, guided by a prime editing guide RNA (pegRNA), to introduce precise edits (insertions, deletions, or base substitutions) without creating DSBs.

      Reduced Off-Target Effects:
      Programmable and Precise: Prime editing requires correct binding of both the Cas9 nickase and the pegRNA, enhancing specificity. No DSBs: Like base editing, prime editing avoids DSBs, reducing the risk of large-scale genomic rearrangements and minimizing unintended indels.

  • Finally, validate predicted off-target sites using sequencing or genome-wide assays to ensure accuracy.
• How do I determine the best CRISPR genome editing system for my application?

  OmniGuide offers four 'standard' gRNA types for popular CRISPR systems: CRISPR/Cas9, prime editing, base editing, and CRISPR/Cas12a, as well as custom gRNA for other editing systems. Each system produces distinct edits with unique pros and cons:

  CRISPR/Cas9: Robust at generating blunt-ended double-strand breaks (DSBs) for knockouts or HDR knock-ins. Extension validation across many cell types, with many derivatives available (dCas9, CRISPRi/a, etc.). However, DSBs can trigger unwanted insertions/deletions or chromosomal rearrangements and sgRNA must be designed carefully to minimize off-target activity.

  Prime editing: Enables small insertions, deletions, and all possible base substitutions without requiring DSBs or donor DNA templates, with lower reliance on error-prone repair mechanisms compared to Cas9. However, prime editing is often less efficient than Cas9 or base editing, especially in primary cells, and the large size of prime editors makes in vivo delivery challenging.

  Base editing: Can directly convert single bases- C to T (cytidine editors) or A to G (adenine editors)- without DSBs, often with higher efficiency than with prime editing and reduced byproducts compared to Cas9 DSBs. However, base editing is limited to these single bases changes, and within a narrow editing window. 'Bystander sites' within the window or at off-target sites can case off-target edits, and the large size of base editors poses delivery challenges similar to prime editing.

  CRISPR/Cas12a (Cpf1): A distinct PAM requirement (TTTV) expands the Cas12a targeting scope compared to Cas9, and the 'stick ends' (staggered cuts) that result can improve certain HDR knock-ins. Cas12a also uses shorter gRNAs (crRNA only), which simplifies design and make synthesis more affordable. However, Cas12a is not as extensively validated as Cas9 in all systems, and editing efficiency relative to Cas9 can vary.

  For other systems or special modifications: You can Book a Call with a CRISPR expert or email us at [email protected] to discuss your specific editing needs.

• What technical support and delivery options are available?

  We have recommended protocols for performing CRISPR knockout experiments available for download in our free CRISPR Ribonucleoprotein (RNP) User Manual, as well as knock-in protocols for some of the mostly commonly used electroporation systems and lipofection reagents.

  OmniGuide RNA can be delivered in either single tubes or 96-well plates, formatted as dry powder or suspended in TE buffer (pH 8.0, 100 µM) or nuclease-free water (100 µM).

• What quality grades does GenScript offer for OmniGuide RNA? How do I decide what grade to use for my CRISPR applications?

  We offer four quality grades of OmniGuide RNA to support different research stages:

  RUO:

  • desalt purified: screening and early-stage research applications
    Advantages: Fast delivery and competitive pricing
  • HPLC purified: validation stage of therapeutic development
    Advantages: ≥90% purity guaranteed for Cas9 sgRNA and Cas12a crRNA. ≥85% purity guaranteed pegRNA and base editing gRNA

  INDEdit: preclinical and IND-enabling studies
  Advantages: Cost-effective with essential QC tests for IND approval. Manufactured at our cGMP facility.
  Compliance: Aligns with FDA and EMA recommendations for preclinical/IND-enabling studies.

  cGMP (clinical grade): clinical studies and commercialization
  Turnaround: 30-day standard delivery
  Purity: ≥80% full-length product (FDA recommended)
  Documentation: Comprehensive IND submission documentation including Drug Master File (DMF)

• In what applications should I consider using base editing or prime editing?

  While far from comprehensive, here are some applications for which you may want to consider using base editing or prime editing:

  Consider base editing for single nucleotide substitutions (C→T or A→G with current editors), such as:

  • Correcting point mutations that cause disease (e.g., a single-base pathogenic variant).
  • Introducing stop codons to knock out genes more precisely than Cas9-induced indels.
  • Studying regulatory variants (e.g., promoter/enhancer SNPs).
  • High-throughput mutagenesis screens where precision and efficiency matter more than broad flexibility.
  • Agricultural improvements via introducing beneficial traits through single base changes

  Consider prime editing for versatile, precise edits beyond simple substitutions, such as:

  • Correcting small insertions/deletions or more complex mutations that base editing cannot address.
  • Introducing specific point mutations when the desired change is outside the editing window or not supported by base editors.
  • Precise knock-ins of short sequences (e.g., tags, restriction sites, epitope sequences) without relying on HDR donor templates.
  • Therapeutic editing research where minimizing indels and off-target effects is critical.
• Does the OmniGuide platform offer arrayed gRNA libraries?

  Yes. Chemically synthesized OmniGuide gRNA can be provided in 96 well plates, with each well containing a custom gRNA targeting a specific gene of interest. Email us at [email protected] to request a quote or order.