(Here we take HIV-1 as an example)

We used a method derived from the method described in the paper by Feng Zhang^{[http://www.nature.com/nbt/journal/v31/n9/abs/nbt.2647.html ZhangFgRNA]}.

Conserved Sequence Analysis

We first tried to extract all conserved regions from the NIH HIV-1 Reference Genome using BioEdit. In this step, we found around 10 alternatives for the next process. Here all screening processes are done in a per-strain basis because of the high mutability of the HIV-1 virus.

Table 1. Base-wise Statistics of One Designed Sequence

As we can see from Table 1, this sequence is highly conserved among about 50% of HIV-1 strains.

Strip out sequences without PAM

Sequences without PAM cannot bind with Cas9 protein and are thus of no use here.

Select gRNA sequences with the best theoretical quality

In this step, we used the tools from Feng Zhang and George Church to analyze off-target activity. Still, we did BLAST ourselves to verify the results.

We have selected gRNA sequences with the best theoretical quality using the experimental formula by Feng Zhang: $$\prod_{e\in{\mathcal{M}}}\left(1-\space W[e]\right)\times\frac{1}{\left(\frac{(19\space-\space\bar{d})}{19}\times 4\space+\space 1\right)}\times\frac{1}{n^2_{mm}} \\ M&space;=&space;[0,0,0.014,0,0,0.395,0.317,0,0.389,0.079,0.445,\\&space;0.508,0.613,0.851,0.732,0.828,0.615,0.804,0.685,0.583]$$

Reference

1. Hsu, P. D., Scott, D. A., Weinstein, J. A., Ran, F. A., Konermann, S., Agarwala, V., ... & Zhang, F. (2013). DNA targeting specificity of RNA-guided Cas9 nucleases. Nature biotechnology, 31(9), 827-832.
2. Mali, P., Yang, L., Esvelt, K. M., Aach, J., Guell, M., DiCarlo, J. E., ... & Church, G. M. (2013). RNA-guided human genome engineering via Cas9. Science, 339(6121), 823-826.