Prime Editing Studio — pegRNA Designer (spacer, PBS, RTT, PE3, twinPE)
Design SpCas9 pegRNAs for any substitution, insertion, deletion or small replacement — spacer, PBS sweep, RTT and 3' extension, PE3 nicking guides, plus twinPE dual-pegRNA design for large edits.
🔒 Nothing you paste is logged or stored
Specify an edit — a substitution, insertion, deletion, or small replacement — on a target region, and get complete SpCas9 pegRNA designs for every usable NGG PAM: the spacer, a primer-binding-site (PBS) length sweep aimed at a ~30 °C melting temperature, the reverse-transcriptase template (RTT) that encodes the edit, and the full 3' extension to append after the sgRNA scaffold, plus PE3 secondary-nick sgRNA suggestions 40–90 bp away on the opposite strand. Designs where the edit itself destroys the pegRNA's own PAM — preventing the edited allele from being re-nicked — are ranked first.
For a substitution or replacement, edit start/end span the region being changed. For a pure insertion, set edit end = edit start − 1. For a deletion, leave the new-bases box empty.
Working on a whole construct rather than one pegRNA design? Open SeqStudio — the full editor, with live feature annotation, plasmid maps, restriction and primer panels, undo/redo, multi-document tabs and GenBank / SnapGene import and export.
How to use the Prime Editing Studio tool
- 1Paste the target region (with flanking sequence around the edit) and specify the edit: start/end positions and the new bases (empty for a deletion; end = start−1 for a pure insertion).
- 2Review the ranked pegRNA designs — spacer+PAM, RTT, the recommended PBS (and the full length sweep), and the ready-to-order 3' extension.
- 3Check whether the edit disrupts the pegRNA's own PAM, pick a PE3 nicking guide if desired, and copy the components — then validate the spacer's genome-wide specificity separately, since off-target activity is not evaluated here.
- 4For a variant library or saturation-mutagenesis campaign, switch to Batch / library mode: one shared target region, then a list of edits (one per line) each designed independently, with a CSV export.
- 5For an edit too large for a single pegRNA's RTT (roughly beyond 40-60 nt), switch to twinPE mode: specify the region to replace and the new sequence, and get a pair of pegRNAs (one per strand) whose 3' flaps share an overlap and anneal to install it without an HDR donor.
Frequently asked questions
Which prime-editing system does this design for?
SpCas9 with the nCas9(H840A)–reverse-transcriptase fusion (PE2/PE3), the dominant architecture. It nicks the PAM-containing strand 3 bp 5' of an NGG PAM, so the tool searches both strands for a nicking guide whose nick sits just upstream of your edit, and builds the PBS/RTT in that strand's frame.
How are PBS and RTT lengths chosen?
The PBS is presented as a full 8–17 nt length sweep with each candidate's melting temperature and GC%, with the length whose Tm is closest to ~30 °C recommended (the widely-used Anzalone 2019 / PrimeDesign heuristic — GC-rich PBS wants to be shorter, AT-rich longer). The RTT covers the edit plus a configurable homology arm (default 13 nt) 3' of it, and the tool flags an RTT whose first reverse-transcribed base is C (associated with lower efficiency).
Does it check off-target activity?
No — there is no reference genome behind this tool, so genome-wide off-target scoring is out of scope. Validate the spacer's specificity with a dedicated off-target tool before ordering.
Which sequence are the reported coordinates measured against?
The target you submitted, before the edit. The protospacer span, the nick position, and the edit start/end are all 1-based inclusive in that pre-edit frame, because Cas9 has to bind the allele you actually have. The exception is a PE3b nicking guide, which only exists once the edit is installed — those are found in the edited sequence, so every nicking guide carries a coordinateFrame field naming which sequence its own coordinates refer to. For an insertion or deletion the two frames drift apart downstream of the edit, so check that field before mapping a guide back onto your template.
Why does ranking prefer edits that disrupt the PAM?
If the edit changes the pegRNA's own protospacer or PAM, the edited allele can no longer be nicked by that same guide, which improves editing outcomes. Designs where this happens are ranked first; when it doesn't, the tool warns you and (for coding regions) points toward adding a silent PAM-blocking change.
Can I design pegRNAs for a whole library of edits at once?
Yes — Batch / library mode takes one shared target region plus a list of edits (label, start, end, inserted bases — one per line), designs each one independently, and lets you export the results (top design per edit) as a CSV. Useful for a saturation-mutagenesis scan or a panel of variants to correct on the same locus.
What is twinPE, and when should I use it instead of single-edit mode?
twinPE (Anzalone et al. 2022) uses TWO pegRNAs nicking opposite strands that flank the region being replaced, each synthesizing a new 3' flap; the two flaps are designed with a shared overlap sequence so they anneal and resolve into the new sequence — no HDR donor needed. A single pegRNA's RTT is practically limited to roughly 40-60 nt, so twinPE is the tool's answer for larger replacements or insertions. Specify the replacement window and the new sequence, and the tool returns independently-ranked left- and right-side pegRNA candidates plus the shared overlap they need to agree on.
Is my sequence stored, and can I use this from code?
No. Your target sequence is used only to compute the designs and is never logged or stored. Both prime_editing_design and prime_editing_twin_design are available via the REST API and MCP server.
More
Guides
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