I need help understanding a figure from a research article. Specifically, Figure

Question

I need help understanding a figure from a research article. Specifically, Figure 2e shown below. I find the text a bit too complicated for me to understand, but I understand the other figure parts. Part A shows an example of the changes they made to the target gene shown in target 1 in part 2b. The darker blue colors signify cleavage, therefore showing that there can be mismatched sequences in the target and the sgRNA will still bind and cause cleavage. In 2C they are testing mismatches within the PAM sequence and if it can still be recognized by the CRISPR complex. For part D it shows the specificity required for the proximal 5'NAG sequence of PAM; with results showing that NAG can also be recognized and indels will occur at a lower expression.

From what I think I know of part 2e is that NRG is referring to the 5'NAG PAM found from the 2d experiment. So I am not sure if I understand correctly; but is the graph showing the amount of NAG sequences in the human genome of how many base pairs away they are from the beginning of the PAM sequence. (so farther distances are more downstream (3' end) I'm guessing).

Reference

Hsu, P. et al. DNA targeting specificity of RNA-guided Cas9 Nucleases. Nature Biotechnology 31, 830 (2013).

Human EMX1 locus 2 kb Target site 1 v PAM GGACATCGATGTCACCTCCA.ATGACTAGGGTGGGCALACCA. CCTGTAGCTACAGTGGAGGETTACTGA TCC CACCCGTTGGT Guide 5 GUCACCUCCAAUGACUAGGG -3' sequence GUCACCUCCAAUGACUAGGN GUCACCUCCAAUGACUAGNG GUCACCUCCAAUGACUANGG GUCACCUCCAAUGACUNGGG GUCACCUCCAAUGACNAGGG GUCACCUCCAAUGRANUAGGG GUCACCUCCAAUIGENCUAGGG GUCACCUCCAAUNACUAGGG GUCACCUCCARANGACUAGGG GUCACCUCCANTUGACUAGGG GUCACCUCCNAUGACUAGGG GUCACCUCNAALIGACUAGGG GUCACCUNCAAUGACUAGGG GUCAC CNCCAAUGACUAGGG GUCACNUCCAAUGACUAGGG GUCANCUCCAAUGACUAGGG GUCNCCUCCAAUGACUAGGG GUNACCUCCAAUGACUAGGG GNCACCUCCAAUGACUAGGG EMX1 target 1 guide sequence G U C A C C U C C A A U G A C U A G G G EMX1 target 2 guide sequence G A C A U C G A U G U C C U C C C C A U EMX1 target 3 guide sequence G A G U C C G A G C A G A A G A A G A A a G EMX1 target 6 guide sequence G C G C C A C C G G U U G A U G U G A U 19 18 17 16 15 14 13 12 11 10 9 8 7 6 5 4 3 2 1

Explanation / Answer

Histogram is used in Figure 2e. Histogram is a graphical representation of continuous quantitative variable. First, the continuous quantitative variable is divided into a number of series of intrevals. Then, depending upon the number of values in each intreval, the height of the bar for that series intreval varies. In generl, the area of the bar represents the frequency and the width of the bar represents class intreval.

Here, in our reference the histogram represents the distribution of NRG PAM occurence in human genome with repect to distance (bp). The distance is divided into number of series of intrevals of serial length 1 bp. The height of the bar in each series intreval represents the frequency of NRG PAM occuence in human genome in that particular intreval.

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