sgRNA Array Designer — Express Many Guides From One Non-Repetitive Cassette
Build a multiplexed CRISPR array that expresses up to 27 sgRNAs from one construct and shares no long stretch with itself — the property that stops it recombining away and getting refused by synthesis vendors.
🔒 Nothing you paste is logged or stored on our servers
Multiplexed CRISPR runs into a problem that has nothing to do with the guides. A Cas9 sgRNA scaffold is about 61 bases of fixed sequence, so expressing twenty sgRNAs from one plasmid the obvious way puts twenty identical copies of it into one molecule, along with twenty copies of the same promoter and terminator. That construct recombines with itself in the cell — losing guides silently, so the screen comes back with a subset working — and synthesis vendors delay or refuse it, because repetition is the largest single cause of synthesis failure. The fix cannot be generated: a scaffold's secondary structure is what Cas9 binds, so randomising it destroys the part. It had to be MEASURED, and it was: twenty-seven scaffold variants that are functionally equivalent and sequence-divergent, published with the method. This tool assembles an array from that collection, selecting promoters, scaffolds and insulators ACROSS all three pools at once rather than within each type — because a promoter and a scaffold can each be clean within their own pool while sharing eighteen bases with each other, which leaves the array exactly as unstable as no selection at all. Then it re-measures the finished molecule, since a repeat spanning the end of one handle and the start of the next promoter exists in neither part. It takes guides and does not choose them: which 20-mer to target, and how well it will work, is a different question with its own tools here.
Paste the protospacers you want to express together to build a non-repetitive array.
How to use the sgRNA Array Designer tool
- 1Check the capacity line first: it says how many sgRNAs fit at each threshold, and that is a property of the parts collection rather than of your guides.
- 2Paste your protospacers, one per line, 20 nt and WITHOUT the PAM — the PAM is in the genome, not in the array.
- 3Pick a maximum shared length. Lower is a more stable array built from fewer available parts; the scaffold pool offers 12 parts at 12 bp, 18 at 15 and 24 at 20.
- 4Read the per-unit table — every unit must show a different promoter and handle — then download the annotated GenBank.
Frequently asked questions
Why can I not just repeat the same scaffold twenty times?
You can build it, and it will often work at first. What happens next is that the twenty identical copies give homologous recombination twenty substrates, so the array loses guides in culture — and because each sgRNA is still expressed from an intact-looking cassette, the screen comes back with some targets repressed and some not, which reads as biology rather than as instability. The same repetition is what makes synthesis vendors delay or refuse the order.
Where do the scaffold variants come from, and can I trust them?
They are from Reis et al., Nature Biotechnology 2019, who measured functional equivalence in cells rather than predicting it — and the parts are used here from the authors' own MIT-licensed release, downloaded and converted rather than retyped. The collection holds 27 scaffolds, which is the hard ceiling on array length; the paper itself demonstrated arrays of 22.
Why does a lower threshold give me fewer sgRNAs?
Because the pools are not mutually non-repetitive at every threshold — selecting from them is the point. At a maximum shared length of 20 bases, 24 of the 27 scaffolds can coexist; at 12 bases only 12 can; at 8 bases, three. You are choosing how much residual similarity to accept, and the tool reports the whole curve so the choice is visible rather than implied.
Does this pick my guides or tell me how well they will work?
No, deliberately. An array of twenty useless guides assembles perfectly, and nothing about the array's sequence says anything about knockdown. Use the gRNA designer and the off-target checker to choose guides — both of those carry their own accuracy statements, because guide activity is a modelled estimate while this assembly is a determinate consequence of its inputs.
What is the insulator for, and can I turn it off?
It is neutral DNA between transcription units, to keep one unit's terminator from interfering with the next unit's promoter. Turning it off removes the spacer pool from the selection, which occasionally lets a longer array fit at a low threshold — the trade is visible in the pool-usage line.
It found repeats in my array anyway. How?
Two ways, and the tool distinguishes them. Either two of your guides are identical or near-identical, which no choice of parts can fix, or a repeat was created by a junction — the end of one handle plus the start of the next promoter — and exists in neither part. That is exactly why the assembled molecule is re-measured rather than only the parts.
More
Related tools
Build a knock-in donor with homology arms around a Cas9 cut site, fold in a PAM-blocking mutation, and get primers that genotype the edit.
Turn one variant description into one oligo order table: the primers that build it and the primers that genotype it.
Design SDM primers from a nucleotide or amino-acid change, QuikChange or Q5 style.