Choose the sgRNA Promoter by Where Transcription Happens

U6 versus T7 promoters for sgRNA expression are a polymerase-and-compartment choice, not a better-guide ranking. Use U6 when the guide must be transcribed inside a mammalian cell by RNA polymerase III. Use T7 when the guide will be made by in vitro transcription, often for later delivery as RNA. PLOS ONE 0148362 states those default jobs. Neither promoter is an edit-rate guarantee.

Pick the Promoter from the Compartment

If the cassette will be transfected or transduced and the cell should make the guide, the default is human U6. If the RNA will be produced in a tube — including RNA prepared for microinjection — the default is T7. The promoter names which polymerase you are recruiting and where that polymerase sits. It does not name cleavage efficiency at your locus.

Chemically synthesized guides are a third path and are out of scope. This page assigns the two promoters in the seed, not a synthesis catalog.

Polymerase, Delivery, and the First Bases

The 5′ start is part of the choice. PLOS ONE: hU6-based vectors require a single G to initiate transcription. RNAs produced from the T7 bacteriophage promoter usually have two G nucleotides, or a G at +1 followed by a purine. Mefferd et al. (2015) restate the U6 rule: transcribed sgRNA sequences must initiate with a G, which can restrict targets or introduce a mismatch to the DNA.

A mismatched start is a transcription constraint. It is not an off-target score. Do not import plant U6 fold-changes from cotton papers into this mammalian/IVT pairing.

A Hybrid Promoter Is an Exception

The same PLOS ONE paper built a U6–T7 hybrid so one vector could serve cell tests and IVT. That is a designed exception for labs that want both jobs on one cassette. It is not a third default column, and the paper's one-locus cleavage numbers are not a universal efficiency law. If you use a hybrid, keep the U6 G in the cognate position. Do not treat the hybrid as proof that editing will be higher everywhere.

What This Page Does Not Redesign

Spacer, scaffold, and terminator layout are cassette anatomy. That page is already written and is not rewritten here. What an off-target is lives on the published off-target definition page. Promoter choice does not replace either job.

After the promoter is chosen, ZettaCRISPR can score guides, and the promoter remains a map annotation in a workspace such as ZettaGene. Those are examples of the next software jobs. They are not a claim that scoring or a map changes which polymerase you recruited.

Promoter Choice Is Not an Edit-Rate Promise

Match polymerase to compartment. Check the first transcribed bases. Stop there. The cited papers document transcription constraints and one-locus tests. They do not license a cleavage-rate, embryo-efficiency, or clinical-safety claim for your target. If the delivery plan is still unnamed, you are not ready to pick U6 or T7.

Frequently Asked Questions

Should I use a U6 or T7 promoter for sgRNA expression?

U6 for in-cell Pol III transcription. T7 for in vitro transcription. The promoter names a polymerase, not an edit rate.

Why does a U6 sgRNA need to start with G?

The U6 promoter initiates at a G. That can restrict spacers or add a 5′ G that is not in the genomic target.

Can one cassette use both U6 and T7?

Yes, as a designed hybrid. PLOS ONE 0148362 built one for cell tests and IVT. That is an exception, not proof of higher editing everywhere.

Does picking U6 or T7 reduce CRISPR off-targets?

Not from promoter choice alone. Off-target meaning lives on the published definition page. This page only assigns the polymerase.