I am trying to knock out a gene involved in virulence from a genomic region of roughly 150kb. So far I have been using CRISPR to target individual candidate genes, but I have recently exhausted my list of candidates without identifying the actual gene, so I have decided to take a different approach.
I plan on using split marker PCR to eliminate chunks of my genome in a stepwise fashion, with the hopes that one of these knockouts will induce a change in virulence, allowing me to focus in on the specific region that was knocked out to find my gene of interest. I understand the process of split-marker PCR, though most of the literature I've read involves using it to knock out specific genes, whereas I will be using it to systematically knock out genomic regions underlying my QTL of interest.
To minimize the number of transformations needed, I'd like to knock out as much of the region as possible in each transformation. The selectable marker I'm using is roughly 3kb, and I've designed my first primers to knockout roughly 5kb, as I've heard of previous successful knockouts of this size. I've also heard that the transformation efficiency decreases proportional to the increase in size disparity between the genomic region knocked out and the selectable marker that replaces it.
I'm currently planning on increasing the size of my knock outs by 2 or 3kb until I fail to get a positive transformant, but I was wondering if anyone has insights or experience with this sort of thing that they could provide me. Or perhaps some molecular theory as to the upper limit of genomic knockouts. I really don't want to do ~30 individual successful transformations to eliminate my full 150kb region. Thanks!
Just to check- did you make sure you knew the copy number the genes you were attempting to knock out and were you able to confirm you knocked out all copies? And do you now whether the genes you're knocking out are essential?
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