Part 4 Design your own sgRNAs

In this last part you can design your own sgRNAs to make a tomato virus-resistant, using CRISPR/Cas9.

Create a flow chart (for your poster) of the steps you go through, and show the positions of the 2 sgRNAs in the sequences. Indicate where the PAM sequences are, and where the complementary sequence of the sgRNA.
Indicate schematically where you expect mutations.

We start from the same gene as J. Chandrasekaran et al. (See part 2), namely the gene eukaryotic translation initiation factor 4Ei, but in tomato.
Find the DNA sequence of this gene in tomato. You can use info on: https://www.ebi.ac.uk/ena/data/view/AY723733.

Questions:

    25. Does this gene have introns?
    26. How long is the gene?
    27. What is the DNA sequence of this gene in tomato?

Open a site for designing sgRNAs: http://crispor.tefor.net/.
Read the text at the top of this site. Then go to Step 1. and copy your DNA sequence from the tomato gene.
The software is looking for sgRNAs, but also tests whether these guides may lead the Cas protein to other places in the tomato genome, which could lead to a cut in an unwanted place, also called off-target. In view of this, the software asks for the organism. Enter this in Step 2 (the scientific name for tomato is Solanum lycopersicum).
Choose at Step 3 the Cas protein that you want to use.

    28. Select SpCas9 here. Why does the software ask for this?
    29. What is the PAM sequence for this Cas protein?

Press SUBMIT.

The software searches for PAM sites. Then it takes the 20 bases prior to that PAM site. The sequence of that 20 bp is the proposed sgRNA.

Then the software will test every combination of sgRNA and PAM-site whether that also occurs in other places in the tomato genome, and may cut there. This search takes some time. If all goes well you will get the results within a minute.

    30. Look at the sequence in the grey box. What is shown here with green, yellow and red letters?

    31. Select two sgRNAs. Motivate why (which criteria) you have chosen these two.

    32. Do you expect off target effects from these sgRNAs? Explain.

    33. At what position (s) of the gene do you expect mutations, if you put both sgRNAs in the plant?

    34. What can happen if both locations are cut at the same time?

    35. Where would you design primers if you later screened for mutations and deletions in a PCR reaction? Identify those primers clearly in the figure with the sequence and sgRNA locations on your poster.