So for this problem, we are to use the given pc FD three vector to express single guide RNA in Drosophila to create a knock out of this N I PP one gene given in the problem statement. So the first part of this problem part a asked us to find the two Pam sites within the sequence. We know from the blurb before this problem that the canonical Pam site is five prime and G g and being any nucleotide. So all we need to do is scan through the sequence to find any NGS. The two that are present in this sequence are near the end. So at the end of the Exxon, we have t oops t g and then in the first entry on, we have another t g. This the first part of the problem also asks us which site we would use to produce a null illegal and why we would prefer that site. So this first Pam site would be the ideal one for us to designer cas nine system around because it would cause a bubble within this Exxon for it to cut. And hopefully we would get a break, causing a frame shift somewhere within this coding sequence and leave us with Leo. The next part of this problem part be asked us to determine the percentage of imprecisely repaired jeans that we could say with confidence would be no Khalil's. So the first possibility is that it prepares exactly where it broke so right at zero and it repairs without any addition. However, this problem statement said that we are only to consider the percentage of the imprecisely repaired genes, so that's eliminated as a possibility. Um, the next option is that it can add up to six nucleotides, however, if it adds three or if it had six. And these could possibly still maintain function because this would cause a frame shift of a full three nucleotides, which would be a single amino acid. Now the same goes for the removal of three or the removal of six um, now that could still maintain some function. Um, it's unlikely, but it could be possible. So now we know that with the addition of 1 to 4 or five and the subtraction of 1 to 4 or five nucleotides, we would have a frame shift that would most certainly disrupt function. So that means we had 12 possibilities. Um, so the addition of six or the subtraction of six nucleotides and then eight, um, known Knowles. So now it is just simple math. 8/12 is equal to 66.6 percent of the imprecisely repair genes would be null alleles. Okay, Next, we are to diagram the cut pc FD three vector, um, and where to ignore the blue segment that would be removed. Um, Now, this would be cut using the BBS one recognition site. So we have our five prime end and three prime ends, so this would be the left side or the orange side. Um, so we've got t t uh, a C and then we've got our matches, and then we've got an overhang, So c a gc. And so that is where it would cut on the left side. On the right side, we would have overhang G two t t. And then we have our pairs, so t a g a key. And here is our overhang. And that would be the vector. So the next part of this problem part D access to design to 24 nuclear tired pieces of D N A that couldn't kneel together and fit inside the cut plasma had, um that would be useful for expressing single guide, aren't it? So, to design a single guide, RNA, um, we have to go back to the blurb before this problem and look at how cas nine bubbles, Um, the genomic target and how single guide RNA fits. So we've got our five prime and three prime here, and the figure looks something like this with our genomic target site, our Pam. And then we've got our complement. And then we've got our single guide RNA that fits here. And so this is our genomic target. So if our single guide RNA is a compliment to the complement of the genomic target, then our 24 um, blip piece of DNA essentially just needs to be the genomic target. Um, what? But we have to include four overhanging pieces. So if you look here, I have already typed it out. But these first and last for our compliments to the cut portion of the plasma, which is C A, g c and G T t t. So we know that that wouldn't heal and then this 24 20 base pair section of D N A. On the top side is simply the last, uh, 20 nucleotides of the coding Exon. And this is because we want the single guide RNA to bubble the genomic, um, genomic target just upstream of the Pam site. And so we have the single guide RNA paired to that region. So next part E asks us to show exactly where cas nine would cut in the n i pp one gene. Um, Now, if we've done our design correctly, then according to the figure before this problem, our CAS nine should cut three base pairs or nucleotides upstream of the Pam. And this is simply shown in the figure before the problem where we have our n g g. And then we've got our bubble and our genomic target site, and they show a base pair another base pair. Excuse me, nucleotide. And then we see after three cast nine cuts. So if we look back to our, uh, and a p p one Jean, um, and we find our and nucleotides towards the end of the Exxon that account for our Pam, we've got a t that you and then we've got the interim with another G. So if we go three nucleotides back from the T, we've got another T A, G and N A. And it would cut between the A and the sea before it, um, specifically between the history and and the Syrian code on So part f of this problem asked us to outline how we would go about making this nor mutation with a component plasma. One common way is to inject a newly fertilized egg. And, um, you would inject it with the plasma expressing the single guide RNA as well as another plasma expressing cas nine. And once they're injected, um, the plasmids are expressed, um, and the cas nine protein and the single guide RNA are expressed. And then if the design process is gone, um, as desired, the genomic target is altered into a no oops. Um, and then you have a fertilized egg with the no alil that will grow into a fully functioning organism Now apart G is very similar. So they asked how you would modify the technique to create a knockin, um, to change the, uh um, the 39 right after the initial initiating met to an Al Ani. Um, now, to do this, essentially, you would just have to create a complimentary piece of D N A. So, um, some sort of piece of DNA that could be expressed on a plasma that is complimentary to the gene. So the a P B one gene, except that in this piece of DNA, you have a few base pairs of overhang on each direction. So essentially you would have something like 80 g representing the Met. Um, and then the A in the 39 would be changed to a. G. Uh huh. And then you could have see t completing the sequence and a pair to make this a complimentary piece of DNA, and then you would need to get this expressed within the cell, like, leave you a plasma. But now, if you have this chunk of DNA expressed, hopefully you would get a, um um ology directed repair instead of No, no, I'm all yes and joining. And if you've done the design correctly, the HDR would lead to uh huh 80 g g c t. In the beginning of the Exxon, which would change the 39 two, uh, excuse me to an l A nine. And that is the end of the problem.