Welcome to the PlatinumCRISPr Help Page
Overview
PlatinumCRISPr supports CRISPR–Cas9 sgRNA design by combining genomic sequence scanning, RNA secondary-structure validation, and efficiency prediction. You may scan genomic DNA or re-test individual guide sequences.
Scanning a genomic sequence
Paste a DNA sequence (up to 2000 bp) or retrieve a gene region from Ensembl. Candidate sgRNAs are identified and evaluated for RNA folding and predicted activity.
Fetching gene sequences from Ensembl
Select an organism and enter a gene symbol to retrieve genomic coordinates and sequence from Ensembl. Retrieved sequences can be scanned directly for sgRNAs.
sgRNA design assumptions
By default, sgRNAs are 20 nucleotides long and start with a G at position 1 to satisfy U6 or U7 promoter requirements. If your experimental design differs, re-test the full sgRNA sequence.
Re-testing a single sgRNA
Enter an sgRNA of 18–23 nucleotides to evaluate RNA folding and efficiency. Even single-nucleotide changes can affect RNA secondary structure.
Interpreting results
Results are displayed using coloured validation boxes. Green indicates correct folding; red indicates incorrect folding. Guides failing structural checks are not recommended.
Structural and sequence features evaluated by PlatinumCRISPr
| Category | Feature | Detailed explanation |
|---|---|---|
| RNA secondary structure | Tetraloop intact | The sgRNA scaffold contains a conserved tetraloop that forms a defined three-dimensional structure required for stable Cas9–sgRNA complex formation. Disruption of the tetraloop, either by misfolding or alternative base pairing, has been shown to strongly reduce or abolish Cas9 cleavage activity. |
| Loop 2 and 3 intact | Stem–loops 2 and 3 are key structural elements of the sgRNA scaffold that mediate direct interactions with Cas9. Correct folding of these loops is required for efficient Cas9 binding and catalytic activity. Misfolding or partial disruption of these loops correlates with reduced editing efficiency. | |
| No bulge mimic | Some sgRNAs form alternative bulge-like structures that superficially resemble the native scaffold bulge but alter the local RNA geometry. These “bulge mimics” can misposition the sgRNA within Cas9, resulting in reduced cleavage despite apparently similar secondary structures. | |
| Not self-complementary | The gRNA sequence should remain largely unpaired and accessible for target recognition, i.e. not be sel-complementary which would enhance pairing within itself. | |
| 51,52,40,41 scaffold pairing | Stabilisation of the RNA-protein complex provided by G62 through non-Watson–Crick hydrogen bonds with A51, A52, and Phe1105, while U63 stabilises the structure via base stacking with A52. The rule tests if these are free to do so and not 'locked' into the secondary structure by complete pairing. | |
| Sequence-based features | GC content | The overall GC content of the sgRNA influences both RNA folding and DNA target binding. Extremely high GC content increases the risk of stable but non-functional RNA structures, while very low GC content can weaken DNA hybridisation, both leading to reduced editing efficiency. |
| Seed composition | The “seed” region of the sgRNA is critical for initial target recognition. Certain nucleotide compositions in this region are associated with more efficient and specific Cas9 binding and cleavage, while others reduce activity or increase off-target effects. | |
| UCYG / CYGR motifs | Short sequence motifs such as UCYG or CYGR have been empirically associated with altered sgRNA performance. These motifs may influence RNA folding, Cas9 interaction, or transcription efficiency, and are therefore evaluated as part of the guide assessment (see Graf et al. ). | |
| UUYY motif | UU-rich sequence motifs (here: UUYY) are associated with an increased risk of premature RNA polymerase III termination when using U6 or U7 promoters, potentially resulting in truncated sgRNAs. |