(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

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: \begin{math} \prod\_{e\in{\mathcal{M}}}\left(1-\space W[e]\right)\times\frac{1}{\left(\frac{(19\space-\space\bar{d})}{19}\times4\space+\space1\right)}\times\frac{1}{n^2\_{mm}} \end{math}