Guide RNA Design Calculator
Guide RNA Design Calculator is a free, scientifically accurate online tool that helps researchers and students design highly efficient and specific CRISPR-Cas9, Cas12a, or base-editor guide RNAs (gRNA) sequences. This calculator uses peer-reviewed scoring algorithms (Doench 2016, Hsu 2013, Moreno-Mateos 2015, CFD specificity score) to predict on-target efficacy, off-target risk, GC content, poly-T termination, self-complementarity, and out-of-frame scores.
About the Guide RNA Design Calculator
The Guide RNA Design Calculator integrates the latest published CRISPR gRNA design rules to deliver reliable predictions without requiring software installation. Whether you are performing gene knockout, activation (CRISPRa), inhibition (CRISPRi), base editing (CBE or ABE), or prime editing, this tool helps you select the best possible 20-nt (Cas9) or 22–24-nt (Cas12a) spacer sequence.
Why Is Proper gRNA Design Critical?
A poorly designed guide RNA can result in:
- Low or no on-target editing efficiency
- High off-target mutations (safety concern)
- False-negative results in functional screens
- Wasted time and expensive reagents
Using a validated Guide RNA Design Calculator dramatically increases success rates and reproducibility.
Scientific Foundations of This Calculator
All calculations are based on peer-reviewed publications:
- On-target score: Doench et al. (2016) – Rule Set 2 & Azimuth 2.0 model (Nature Biotechnology)
- Off-target CFD score: Doench et al. (2016) – Cutting Frequency Determination
- Specificity score: Hsu et al. (2013) MIT algorithm
- GC content & poly-T rules: Moreno-Mateos et al. (2015)
- Self-complementarity & secondary structure: Verified folding energy thresholds
When Should You Use This Guide RNA Design Calculator?
Use it whenever you are:
- Designing gRNAs for mammalian, plant, bacterial, or zebrafish genomes
- Performing CRISPR screens (genome-wide or focused)
- Developing base editors or prime editors
- Optimizing gRNA for in vivo therapeutic applications
- Teaching CRISPR technology in academic courses
User Guidelines – How to Get the Best Results
- Paste your target DNA sequence (with PAM site) – include at least 50 bp upstream and downstream.
- Select your Cas protein (SpCas9, SaCas9, Cas12a, enAsCas12a, etc.).
- Optionally enter up to 3 known off-target sequences with mismatches.
- Click “Calculate” – results appear instantly.
- A score > 70 is excellent, 50–70 is good, < 50 needs redesign.
Detailed Explanation of Output Parameters
| Parameter | Ideal Range | Explanation |
|---|---|---|
| On-Target Efficacy Score | 70–100 | Predicts editing efficiency (Doench 2016) |
| Specificity Score | 80–100 | Lower off-target risk (Hsu 2013) |
| CFD Off-Target Score | < 0.2 per site | Cutting Frequency Determination – lower = safer |
| GC Content | 40–70% | Extreme GC reduces efficiency |
| Poly-T Count | ≤ 3 | ≥4 Ts triggers Pol III termination |
| Out-of-Frame Score | > 65 | Higher = better knockout via frameshift |
This Guide RNA Design Calculator is proudly powered by open science and used by thousands of researchers worldwide. For agriculture-focused CRISPR applications, visit our partner site Agri Care Hub. For in-depth background, read the CRISPR - Wikipedia article.
