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I have a hard time understanding what this paper is talking about when it says:

We observed maximal cleavage at sites oriented tail-to-tail and separated by -10 bp to +30 bp (Fig. 2d). Finally, adjacent sites on the same DNA strand (head-to-tail orientation) did not show damage by NHEJ

Then it shows a figure/chart that I don't understand. I don't know the head and tail or the gene is, in terms of what it is relative or with respect to.

Any help would be appreciated. Thanks.

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The heads and tails, in this paper, refer to the orientation of sgRNA binding sites. If there are two tandem sites in the same orientation then they are referred to as head-to-tail (end of the first site followed by the beginning of the second site). It is also apparent from the excerpt that you have included in your question.

Head-to-head and tail-to-tail refer to the site pairs that are placed in the opposite strands; the former (H2H) refers to a configuration in which the beginnings of the two sites are closer whereas the latter (T2T) refers to the case where the ends of the two sites are closer.


Regarding your question about the placement of sites within -10 to +30 bp of each other:

I think the point of the paper is that the two cuts by the nickases should be close enough and in the T2T orientation for a maximally disruptive double strand break. If the distance between the single stranded cuts (nicks) is increased then they effectively are just two single strand breaks which are repaired efficiently. The authors also conclude that using two nickases in this fashion produces has lower chances of mistargeting compared to that of a normal Cas9 (that makes double strand cuts).

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