Assume we do have a double stranded DNA sequence in human cells, say
...ATCGATATCGATATTGCAGAGCATAGCTATAA...
...TAGCTATAGCTATAAGCTCTCGTATCGATATT...
Now I want to cleave one strand, such that I do have
...ATC TAA...
...TAGCTATAGCTATAAGCTCTCGTATCGATATT...
(I hope it is represented correctly here.)
The distance is not so important to me, so it can be 20 bp I remove or 1000s. Hence, if I need some PAM or so and it is not nearby the target deletion site, I will simply move in 5' resp. 3' direction until I find it and cleave there, as long it is ensured that I have the target site single stranded then.
Another thing that would be sufficient for my project would be that on a specific site the DNA gets unwinded in vivo (perhaps by some CAS enzyme like dCas9, though others might be better suited here). Then one of the strands might be "hidden" by the machinery, but the other one should be accessible to toy around with it. Is such a thing possible?
I would be very grateful I you could help me here!
This would be a kind of difficulty to be realized in vivo. It is possible that you design two nicks in the same strand. However, when the nicks are more than 6 to 7 bases away from each other, the nicked small fragment wont get off from the DNA molecule. The nicking will be repaired in vivo in a pretty short time.
Dear Ziguo, many thanks for your reply. Regarding the second question, where I try to have a large strand of ssDNA, could I fuse dCAS9 to a helicase, such that the dCAS9 bind in sequence-specific manner to the dsDNA, opens the DNA and the helicase opens it for, say several tens of bps. Is that realistic?
If you use dCas9 do not expect cleavage activity. dCas9 can be used as a gene silencing or activation tool.
If you need KO, KI, Multiple or Single Point Mutations then use regular cas9 or Cas9 HF. The question is what is your aim? If you want to have ssDNA (>250 bp) then you need to read this journal : doi: 10.1371/journal.pone.0094752
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