We propose a web application for the Design and Optimization (CRISPR-DO) of guide sequences in several genomes (human, mouse, fly, worm and zebrafish). CRISPR-DO integrates an sgRNA efficiency prediction model (Xu, et al., 2015) and an off-target scoring function (Hsu, et al., 2013), which allow the users to evaluate the “goodness” of an sgRNA in both sensitivity and specificity. we annotate each target sequence with the PhastCons conservation score as well as the overlaps with exons, DNase I hypersensitive sites (DHSs), and SNPs for better functional characterization. We also integrated our target sequence search result with the powerful WashU Epigenome Browser (Zhou, et al., 2011) to facilitate the visualization and selection of target sequences.
CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats / CRISPR-associated protein 9) originally came from bacterial host defense and has given scientists new insight into site-specific genome editing. The CRISPR/Cas9 technology requires an sgRNA with a ~20bp guide sequence to pair with the target DNA, which enables the Cas9 protein loading to the correct location and introduces a DNA double-strand break (DSB) (Cho, et al., 2013; Cong, et al., 2013; Jinek, et al., 2012; Mali, et al., 2013).
This technology requires a tracrRNA:crRNA duplex with a ~20bp guide sequence to pair with the target DNA sequence. This enables the Cas9 protein binding and introduce a double-strand break in the DNA, inducing insertion or deletion mutations (indels) or precise alterations through HR(homologous recombination) or NHEJ(non-homologous end joining). The tracrRNA:crRNA deplex was also engineered into a single guide RNA (sgRNA) with a 5' side DNA target region (spacer) and the 3' side Cas9 binding region. The protospacer adjacent motif (PAM) at the end of spacer is also a necessary component for the recognization of DNA target.