CRISPR SpCas9 NGG PAM and Guide Sequence Inspection Tool
Inspect candidate SpCas9 sites in an 80-base pUC19 reference window and edit the supplied sequence to see the existing finder recalculate. This engine recognises a 20-base protospacer followed by NGG on either strand. It does not implement Cas12a TTTV recognition or predict on-target activity and genome-wide off-target specificity.
The same cited short window used by the existing SpCas9 worked example. It is treated as a linear sequence window rather than a complete circular vector or genomic locus. Candidate coordinates and exact-repeat counts apply only to the supplied sequence.
Read the PAM in the correct orientation
The PAM is part of the target DNA context recognised by the nuclease, not simply an extra motif appended to an arbitrary guide sequence. The finder scans each strand under the same directional rule. A reverse-strand candidate therefore requires careful interpretation of both its displayed coordinates and its own five-prime-to-three-prime sequence.
The 80-base example is deliberately a local inspection exercise. It contains no claim about a chromosomal target or a complete off-target search space. Editing the sequence changes the local candidate list and can demonstrate how a PAM mutation removes or creates a candidate. That numerical result remains tied to the entered window and the implemented SpCas9 rule.
Separate enumeration from guide qualification
The existing finder reports sequence properties such as GC fraction, a longest homopolymer run and exact repeats in the supplied sequence. These are descriptions that may inform a review; they are not calibrated activity or specificity scores. Do not combine them into an invented ranking or treat the first listed candidate as a recommended experimental guide.
Before an experiment, use the actual organism and genome build, the intended nuclease, complete target context and an appropriate qualification workflow. Variant alleles, paralogues, delivery and cell-specific biology can matter. The local inspector helps check sequence orientation and the presence of a canonical PAM, while those additional analyses address risks and performance that a short pasted sequence cannot resolve.
Equations and variables
Implemented SpCas9 site pattern
5′–N20–NGG–3′
- N20
- Twenty-base protospacer on the inspected strand
- NGG
- Three-base PAM on that strand
- N
- Any unambiguous DNA base accepted at that position
GC fraction
GC (%) = 100 × (nG + nC)/ndenominator
- nG, nC
- Counts of guanine and cytosine
- ndenominator
- Length on the alphabet/ambiguity policy stated by the calculation
Scope and limitations
- SpCas9 NGG only: Cas12a TTTV, alternative Cas variants and permissive PAM models are not implemented.
- Other-occurrence counts are exact matches within the supplied sequence, on both strands. They are not a mismatch-tolerant genome search or an off-target assessment.
- The short-window inspector is bounded to manageable sequence lengths. A missing site near a linear boundary may require additional flanking sequence.
Sources and provenance
- GenBank pUC19 record L09137.2Existing source notice: NCBI public domain
Versioned sequence and SHA-256 are pinned in the shipped example.
- Jinek et al.: programmable dual-RNA-guided endonucleaseArticle citation; no experimental data reproduced
- Zetsche et al.: Cpf1 as a distinct class 2 CRISPR endonucleaseArticle citation; no experimental data reproduced
Explains why the absent Cas12a mechanism cannot be represented by relabelling an NGG finder.
Technical questions
Can this page find Cas12a TTTV sites?
No. The current engine explicitly implements SpCas9 with a twenty-base protospacer and NGG PAM. Cas12a has different recognition and orientation requirements. Mentioning TTTV in a methodology comparison does not add that capability to the finder.
Does zero other occurrences mean a guide is specific?
No. The count examines exact protospacer repeats only within the supplied window. It does not search the rest of a genome or allow mismatches and bulges. A specificity statement needs a dedicated search against the relevant reference and a clearly stated model.
Why are guides listed on both strands?
Each strand can contain a protospacer followed by the required PAM in its own five-prime-to-three-prime direction. The displayed coordinates map those candidates back to the input. Keep the strand and PAM together when interpreting or copying a candidate.
Connected workflows
- Primer-pair Tm and template matching
Compare guide orientation with another template-bound oligonucleotide workflow.
- Six-frame translation and ORF inspection
Inspect the sequence context around a candidate site.
- GenBank and FASTA plasmid mapping
Retain the accession and topology of a larger construct.