CRISPR/Cas9 is an adaptive immunity system in bacteria and most archaea (Koonin and Makarova, 2009; Horvath and Barrangou, 2010). The CRISPR/Cas9 gene editing system is comprised of two key components, a small guide RNA (gRNA) and a Cas9 endonuclease (Deltcheva et al., 2011; Jinek et al., 2012). The gRNA is a chimeric RNA molecule of tracrRNA and crRNA (Ran et al., 2013) which guides the Cas9 protein to the target site in the genome. Therefore, selecting a target site with high on-target activity and low off-target effect is crucial for gene editing. Previously, we have discovered that the gene editing activity of CRISPR-Cas9 in mammalian cells was affected by several factors, such as the secondary structure and chromatin accessibility of the guide sequences (Jensen et al., 2017). Strikingly, previous results from us and other research groups consistently revealed that the CRISPR gRNA activities are highly variable. Thus, several in silico gRNA design web tools and algorithms have been developed to facilitate CRISPR design and applications.