CRISPR gRNA Design Rules
A good CRISPR guide RNA (gRNA) is a short spacer that sits next to a valid PAM, has moderate GC content, avoids sequences that break transcription or synthesis, and is unique in its target genome. The rules below match the transparent heuristics SeqBench's designer applies — use them to shortlist candidates before checking off-targets in a genome-aware tool.
| Parameter | Recommendation | Why |
|---|---|---|
| Spacer length | 20 nt (SpCas9); 21 nt (SaCas9); 23 nt (Cas12a) | Matches the nuclease's natural protospacer length |
| GC content | 40–70% ideal (30–80% usable) | Very low/high GC reduces activity and stability |
| Poly-T (TTTT) | Avoid | A run of ≥4 T's terminates Pol III (U6/H1) transcription of the guide |
| Homopolymer runs | Avoid runs ≥5 of any base | Hurt specificity and oligo synthesis quality |
| 5′ nucleotide | Prefer/append a G when using a U6 promoter | Pol III initiates efficiently from a 5′ G |
| PAM | Required, adjacent to the spacer (see PAM table) | No PAM, no cleavage; not part of the spacer you clone |
| Cut site | SpCas9: ~3 bp 5′ of the PAM (blunt); Cas12a: staggered, distal to PAM | Determines where the edit lands |
| Seed region | ~10–12 nt PAM-proximal — keep it unique | Mismatches here most strongly block off-target (and on-target) cutting |
| Off-target | Check genome-wide; minimise matches, especially in the seed | Specificity is genome-dependent and can't be judged from the guide alone |
Values are practical defaults — on-target efficiency and off-target risk vary by nuclease, locus and genome, so verify for your system.
Spacer length and PAM
Start by picking the nuclease, because it fixes the spacer length: 20 nt for SpCas9, 21 nt for SaCas9 and 23 nt for Cas12a. The spacer is the genomic sequence immediately adjacent to a valid PAM — see the CRISPR PAM table for each enzyme's PAM. Crucially, the PAM is not cloned into the guide: it must be present in the target DNA, but the spacer you order or express stops at the edge of the PAM.
Sequence rules (GC, poly-T, homopolymers, 5′ G)
Keep GC content in the 40–70% sweet spot (30–80% is usable); guides far outside this band tend to be less active or less stable. Avoid a poly-T runof four or more T's (TTTT): it is a Pol III terminator, so a U6- or H1-driven guide carrying it gets truncated during transcription. Likewise avoid homopolymer runs of five or more of any base, which hurt specificity and degrade oligo synthesis quality. When using a U6 promoter, prefer a guide that starts with G, or append a 5′ G, because Pol III initiates efficiently from a 5′ G.
Seed region and specificity
Not all positions in the spacer matter equally. The seed region — roughly the 10–12 nt closest to the PAM (PAM-proximal) — dominates target recognition. Mismatches in the seed most strongly block cutting, for both on-target and off-target sites, while mismatches in the PAM-distal end are better tolerated. A guide whose seed is unique in the genome is far less likely to drive off-target cleavage.
Choosing the target site
For a knockout, target an early, constitutive coding exon or a known functional domain so that an indel disrupts the protein in every transcript. Avoid the extreme 3′ end of the coding sequence, where a frameshift may leave most of the protein intact. Also avoid placing the spacer or PAM over a common SNP: a polymorphism in your samples can abolish targeting in some alleles or individuals.
Off-target checking
Off-target risk is genome-dependent and cannot be judged from the guide sequence on its own. Use the rules above to shortlist candidates, then verify each one in a genome-aware specificity tool before ordering oligos, paying special attention to near-matches in the seed region. Note that on-target efficiency models (for example Doench 2016) require a trained model rather than a simple heuristic, so treat any rule-based ranking as a first pass, not a final score.
Frequently asked questions
- How long should a gRNA spacer be?
- It depends on the nuclease: 20 nt for SpCas9, 21 nt for SaCas9 and 23 nt for Cas12a. Each value matches the natural protospacer length that nuclease recognises. The PAM is adjacent to the spacer but is not part of the spacer you clone.
- What GC content should a guide RNA have?
- Aim for 40–70%, with 30–80% still usable. Very low or very high GC reduces guide activity and stability, so guides outside the usable band are best avoided when alternatives exist.
- Why should I avoid TTTT in a guide?
- A run of four or more T's (a poly-T stretch) acts as a Pol III terminator. When the guide is expressed from a U6 or H1 promoter, that signal cuts transcription short and you lose a functional guide.
- What is the seed region of a gRNA?
- The seed region is roughly the 10–12 nucleotides closest to the PAM (PAM-proximal). Mismatches here most strongly block cutting, so the seed dominates target recognition — keep it unique in the genome to limit off-target activity.
- How do I check a guide for off-targets?
- Off-target risk is genome-dependent and can't be judged from the guide sequence alone. Shortlist candidates using the rules here, then verify each one in a genome-aware specificity tool before ordering oligos.
- Do I need a 5′ G on my guide?
- When expressing the guide from a U6 promoter, a 5′ G helps Pol III initiate efficiently. If your spacer does not already start with G, you can prefer a guide that does or append an extra G at the 5′ end.
See also
Related tools and references
Use these related pages when this table raises a practical calculation or workflow question.