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Short answer: sgRNA design means choosing a roughly 20-nucleotide targeting sequence (the spacer) that is unique in the genome and sits immediately next to a PAM, then checking how likely that guide is to cut elsewhere. For Streptococcus pyogenes Cas9 the PAM is NGG. Design tools score candidates for on-target activity and off-target risk; the score ranks options, it does not guarantee an outcome. This page is general laboratory information, not a protocol or medical advice.
What is CRISPR-Cas9?
Engineered CRISPR genome editing has two working parts: a Cas enzyme that cuts DNA and a guide RNA that tells it where to cut (Addgene CRISPR Guide, read 2026-11-07). Other Cas enzymes use other PAM sequences and recognise targets differently; see Cas12a and Cas13, and for transcriptional control without cutting, CRISPRa. For the trade-offs of the technology as a whole, see CRISPR pros and cons.
What is the difference between gRNA and sgRNA?
In practice the two terms are used interchangeably. Addgene describes the guide RNA as a synthetic fusion of the bacterial crRNA and tracrRNA that gives targeting specificity and binds Cas9, and notes it is called gRNA or sgRNA. “Single guide” refers to the fusion: the two natural RNAs joined into one molecule.
| Term | What it is (per Addgene, 2026-11-07) |
|---|---|
| gRNA / sgRNA | The guide RNA that directs Cas9; the engineered fusion of crRNA and tracrRNA |
| Spacer (targeting sequence) | User-defined sequence of about 20 nucleotides that sets the target; it is the 20 nt preceding the PAM and excludes the PAM and the scaffold |
| Scaffold | The part of the guide that binds the Cas enzyme |
| PAM | The short motif the target must sit immediately next to; NGG for SpCas9. It lies in the genomic DNA, not in the guide |
How to design a CRISPR guide RNA
The criteria below come from Addgene’s CRISPR guide (read 2026-11-07). Online design tools apply them automatically, so the checklist is mainly for understanding what a tool is doing and for reviewing its output.
- Pick the Cas enzyme first. The enzyme fixes the PAM, and the PAM restricts which sites can be targeted.
- Find sites next to a PAM. The target must sit immediately next to a PAM (NGG for SpCas9).
- Make the spacer about 20 nt and take it from the sequence preceding the PAM. Do not include the PAM in the guide.
- Require a unique target. The target sequence should be unique in the genome.
- Weigh the seed region. The 8-10 bases at the PAM-proximal 3′ end must match; mismatches there block cleavage, whereas mismatches distal to the PAM (the 5′ end) can still allow cleavage.
- Screen for off-targets. Most guides have partial matches elsewhere in the genome, so candidate off-target sites need to be listed and compared.
On-target and off-target scoring, and design tools
Design tools rank candidates with an on-target activity prediction and an off-target estimate. The anchor reference is Doench et al. 2016 (Nature Biotechnology 34(2):184-191, “Optimized sgRNA design to maximize activity and minimize off-target effects of CRISPR-Cas9”). Its abstract states that on-target activity and off-target effects of individual sgRNAs can vary widely; the authors derived design rules from genome-wide human and mouse library screens, profiled the off-target activity of thousands of sgRNAs, and developed a metric to predict off-target sites.
What a score does and does not mean:
- A score is a prediction trained on particular screens and cell contexts. Because activity “can vary widely”, a high-ranked guide can still underperform and is normally tested.
- Different tools use different models and versions. When you report a design, record the tool, its version and the genome build used.
- A low off-target estimate is not proof of no off-target editing; it is a ranking aid.
Addgene recommends using online gRNA design tools and notes that library screens are designed for high on-target and low off-target activity. For wider sequence-analysis software, see Geneious Prime; for where plasmids and guide constructs are shared, see Addgene; for a commercial CRISPR supplier, Synthego.
Off-target mitigation options
| Option | What Addgene says (2026-11-07) |
|---|---|
| Nickase Cas9 (Cas9n) guide pairs | Two nicks on opposite strands are needed to make a double-strand break, which lowers off-target editing |
| High-fidelity variants: eSpCas9(1.1), SpCas9-HF1, HypaCas9 | Reduce off-target editing compared with wild-type Cas9 |
| Truncated gRNAs, Sniper-Cas9 | Mentioned as approaches for improving specificity |
Common misunderstandings
- “The PAM is part of the guide.” It is not: the targeting sequence excludes the PAM and the scaffold.
- “gRNA and sgRNA are different reagents.” For Cas9 work they name the same engineered guide.
- “Any 20-mer will do.” The site must be adjacent to a PAM and unique, and activity varies widely between guides.
- “A mismatch anywhere stops cutting.” Seed-region mismatches block cleavage; distal ones often do not.
FAQ
How to design CRISPR guide RNA?
Choose a Cas enzyme, find a unique target of about 20 nt immediately next to its PAM, check the PAM-proximal seed region, and screen for off-target sites, usually with an online design tool. See the checklist above.
What is the difference between gRNA and sgRNA?
The terms are used interchangeably for the engineered, single-molecule guide RNA that fuses crRNA and tracrRNA.
Is CRISPR-Cas9 illegal?
Using CRISPR-Cas9 in the laboratory is not illegal as such; legality depends on the application and the country, with the strictest rules concerning heritable human germline editing. Country-by-country detail is in CRISPR germline editing: legal status by country.
What is CRISPR-Cas9?
A genome-editing system in which a guide RNA directs the Cas9 enzyme to a matching DNA site next to a PAM, where Cas9 cuts.
Sources
- Addgene, “CRISPR Guide”, https://www.addgene.org/guides/crispr/ (read 2026-11-07).
- Doench JG, et al. “Optimized sgRNA design to maximize activity and minimize off-target effects of CRISPR-Cas9.” Nat Biotechnol 2016;34(2):184-191 (abstract read 2026-11-07).
