siRNA/shRNA Design Tool

Enter Query Sequence


Enter Sequence(s) using a method below (Plain for FASTA format).

No sequence selected. Please enter at least one sequence.



siSPOTR is a RNAi design tool that produces candidate siRNAs and shRNA with the lowest off-targeting potential for a given target transcript. Instructions


  1. Input sequence(s) for siRNA design. Click the appropriate button to paste or upload the sequence(s). Single sequences can be in plain or FASTA format. Multiple sequences require FASTA formatting.
  2. Select the number of candidates desired in the output.
  3. (Advanced) Modify input parameters:
    • GC Content (%Min/Max): Filters candidate sequences based on specified GC content range.
    • 5' Passenger G/C Number: Forces the passenger strand to have high 5' end stability. This decreases likelihood of loading the unintended (passenger) strand. Additional parameters to promote guide strand loading are automatically included in the design.
    • Pol III Expression: Removes sequences with ≥4 T/U's. Stretches of 4 or more U's can serve as a termination signal for RNA Pol III. Since many shRNA/artificial miRNA expression vectors use Pol III promoters (e.g. U6, H1) to drive expression, these sequences should be avoided to prevent pre-mature transcript termination.
  4. Run siSPOTR
  5. Browse additional off-target information (Highly Recommended): Click "Off-Target Details" to:
    • BLAST against transcript databases
    • View the overall number of off-targets and the distribution of more potent seed site-types (7mer-1A, 7mer-M8, 8mer)
    • View/download list of all predicted off-targets with site-type information