pirScan is a tool that predicts the presence of piRNA targeting sites within a given sequence and suggests silent mutations that can be introduced to avoid silencing of transgenes by piRNAs. The user can input a mature mRNA or spliced DNA sequence in plain text format and be provided with a concise graphical representation of which sites within the sequence display predicted piRNA targeting sites. The user can then choose between suggested silent mutations that will allow the input sequence to escape predicted piRNA targeting, but still retain the same amino acid sequence.
For each piRNA targeting site, pirScan lists the types and positions of mismatches between a predicted endogenous piRNA and the user’s input sequence. The pairing between the 21 nt long piRNA and the corresponding position in the input sequence is displayed for clarity, with the input sequence listed 5’ to 3’. The target sites within the input sequence can be downloaded as a .csv table or as a graphic to be displayed or manipulated by the user outside of our provided framework.
A user can subsequently change his/her input sequence by following our intuitive graphical interface. Proposed silent mutations will be displayed which can be chosen by the user. Each proposed mutation also indicates which targeting rule(s) will be broken if the nucleotide is changed. The proposed changes are ranked so that mutations which break the most important rules (i.e. mismatches within the seed region) will be listed first and selected by default. If more than one piRNA targeting site were present in the input sequence, then each piRNA will have its own separate table complete with the possible silent mutations to introduce. Each table is displayed according to the 5’ to 3’ order which each piRNA is predicted to interact with the input sequence. Upon the user’s completion of the proposed mutagenesis, the modified sequence can be immediately re-submitted for scanning to verify that the sequence will successfully escape piRNA targeting and ensure that new targeting sites were not inadvertently introduced. If pirScan still predicts piRNA targeting sites in the modified sequence, the suggested changes for these sites are presented for further modification. Once the user is satisfied with the adjusted input sequence, the modified version can be downloaded as a text file. Additionally, a table and graphic which highlights the user-selected mutations are available for export.