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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.
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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.