1. What results does the automated CRISPR planner provide?
    Once the crRNA is chosen by the user, the algorithm will try and introduce the relevant strand with codon mutations (to alter the amino acid) and mutations to prevent reattachment in the relevant section (PAM site/crRNA) that will also enable the insertion of a new restriction site.
    If a new restriction site cannot be inserted using the two types of mutations mentioned above, the algorithm will try to add point mutations to insert a new restriction site.
    In case of failure, the algorithm will try to choose the codon and prevent-reattachment point mutations to enable the removal of an existing restriction site, and if not successful, the algorithm will try to add point mutations to remove an existing restriction site.
    If all steps have failed, the tool will provide the user with a message stating that the insertion/removal of a restriction site was not possible.
    However, when results are achieved, each result will specify the codon point mutations, the prevent-reattachment point mutation and the restriction site point mutation (if necessary), along with the restriction site inserted/removed and the repair template (consists of two 35 nucleotide homology arms on each side + section with desired edit).
    Each point mutation will be represented by the index of the nucleotide, the former nucleotide and the new nucleotide. The relevant ssODN strand (sense or anti-sense) will be stated.

  2. How does providing preferred restriction enzymes to the Automated CRISPR Planner affect the algorithm?
    When providing the tool with preferred restriction enzymes, it aims to find CRISPR plans that use one of the given enzymes.
    The algorithm will only search for restriction sites using the enzymes you provided it.
    If you wish to search results using all restriction enzymes known to the system, just leave the box empty.

  3. What does it mean if the query takes too long to return?
    In this case, there are probably some difficulties in finding a restriction enzyme to insert.
    If you specified the restriction enzymes you wish to work with, try extending that list or remove it altogether to search all restriction enzymes known to the program.
    Also, if you specified a high number of results you wish to get, try lowering that number to check whether the algorithm has come up with some results.

  4. Where can I find a full list of the restriction enzymes the algorithm searches for?
    Our algorithm uses the New England Bio Labs's list of restriction enzymes: New England BioLabs.
    If you wish to add a restriction enzyme that is not included in this list, please contact us.

  5. What would happen if I specified a restriction enzyme's name that is unknown to the algorithm?
    The algorithm would disregard it.
    Your results show which of the enzymes you entered are valid and were used in the search.

  6. In what format should I enter the restriction enzymes I wish to use?
    The algorithm will accept a list of names of the restriction enzymes you are interested in, without the HF notation for high fidelity.
    The enzymes' names can be separated by commas, tabs, semi colons or spaces.
    To check whether your restriction enzymes' names are correctly formatted, check your results.

  7. Could there be other CRISPR designs that the algorithm didn't find?
    Yes. The algorithm's purpose is not to find all possible designs for the point mutation you are interested in, but to find the best options relevant for your query and return them when the requested amount of possible results is achieved (default of one result).
    If you are not satisfied with the results given, you can alter the maximum results box to be given more results.
    However, if you are given less results than you specified in the maximum results box, then the algorithm could not find more results, thus changing the bar of maximum results won't make any difference.
    If you feel something is wrong with the results, please contact us with the details of your query.

  8. Why is providing the nucleotide sequence optional? When is it necessary to provide it?
    The algorithm will try to extract the nucleotide sequence (unspliced + UTR) from several sources.
    However, in case the algorithm cannot find the relevant sequence (or is faulty and doesn't match the protein sequence it later extracts), an error would be presented to the user, specifying that the system's findings do not match the amino acid you wish to alter.
    In that case, you have the possibility to provide the correct sequence yourself.

  9. Are the restriction sites found filtered? How are they filtered?
    Yes. The restriction sites found in the process of inserting/removing restriction sites are filtered out if a same site is found in a distance of less than 150 nt from the found site.
    Restriction sites that pass the filtration are also sorted by the proximity to the Double Strand Break (DSB) point, by distance from other same sites in the range of 500 nt around the found site and by the frequency of that site in the range of 500 nt around the found site.

  10. If the codon we wish to mutate has point mutations before and after the DSB, which arm would be the one we mutate?
    If the number of point mutations on one side of the DSB is higher than the number of point mutations on another, then the side with the higher number of mutations will be chosen as the section to be mutated.
    If not, the algorithm first defines the sequence with the PAM site to be the mutated sequence, and if mutating the PAM sites is impossible, the algorithm will take the second sequence to design the CRISPR plan onto.

  11. How far away from the DSB point would the algorithm try to insert mutation (or: what range is taken to be the mutation zone)?
    The mutation zone is defined to be ranging from the minimum between the lowest point mutation introduced to the strand among the point mutations that change to amino acid and mutate the reattachment section (PAM, crRNA) and DSB-20, and to DSB-1.

  12. What is the numbering scheme the indices are represented by?
    The results incorporate zero-based numbering; the initial element of a sequence is assigned the index 0.

  13. What protocol does the automated CRISPR planner follow?
    Our algorithm is based on steps and the scheme described in this paper:
    Paix A, Folkmann A, Seydoux G. Precision genome editing using CRISPR-Cas9 and linear repair templates in C. elegans. Methods. 2017;121-122:86-93. doi:10.1016/j.ymeth.2017.03.023
    Our lab has made a few alterations in the CRISPR protocol, you can view our revised protocol here:
    CRISPR protocol alterations and tips