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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...

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.

I would be very grateful I you could help me here!

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    $\begingroup$ I don't understand what your question is - why are you trying to remove a single strand in vivo? What is PAM in this context? Please edit your question to add additional explanations and details. $\endgroup$ – MattDMo May 11 '16 at 0:51
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Both TALEN and ZFN gene editing technologies use a pair of single-strand endonucleases that ultimately lead to a double strand break. Cas9 in CRISPR-Cas technology, however, cuts both the DNA strands (there are some Cas enzymes in certain bacterial species that cut only a single strand [ref]).

     enter image description here


Image courtesy: http://www.xenbase.org/other/static/CRISPr.jsp

So, if you use a single unit of the pair, you can cut one strand of the DNA. You can use another nuclease to impart a cut at another site in the same strand. This will still not lead to the removal of the DNA sequence between the two cut sites. There is no available technology that can do this in-vivo. Possibly (a wild guess), you can use a modified oligonucleotide (or even an IVT synthesized RNA) that can bind to this stretch of sequence and break its interaction with the other DNA strand. In any case, a stretch of ssDNA would not be stable and be repaired quickly by polymerase.

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  • $\begingroup$ Many thanks for your reply. 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? $\endgroup$ – tobias May 11 '16 at 8:11
  • $\begingroup$ @tobias Possibilities are infinite but we should go with the one that is simplest in principle and is easy to implement. $\endgroup$ – WYSIWYG May 11 '16 at 8:29
  • $\begingroup$ Sure :). What would you suggest for it? $\endgroup$ – tobias May 11 '16 at 10:06
  • $\begingroup$ @tobias I don't think such a protein has been engineered yet. That would be a research project in itself and you would have to do many experimental try-outs. So, I cannot suggest much regarding that- you would have to try it. What I said in the answer (the guess) would be the first thing that I would try because it is easy. $\endgroup$ – WYSIWYG May 11 '16 at 12:12
  • $\begingroup$ 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? $\endgroup$ – tobias May 11 '16 at 19:54

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