FIGURE 1.

Design principle of a CRISPR/Cas9 expression vector for construction of large-scale libraries. The vector requires two minimal components, i.e., the single-guide RNA (sgRNA) sequence and the Cas9 gene that can both be expressed from a single-vector system. The sgRNA is composed of a variable crRNA and a constant tracrRNA. The gene sequence can be inserted by a simple adaptor cloning step in the 20 bp crRNA region. Furthermore, multiple target sequences of the same gene can be inserted to increase efficiency and reduce off-target effects. At the 3′-end of the crRNA sequence, a 2 bp (base pair) protospacer adjacent motif (PAM – green box) of the sequence GG is essential, but AG may be used to a lesser extend as well. A 12 bp seed region (blue box) at the 3′-end is required for sequence tar-geting with additional 8 bp at the 5′-end contributing to specificity (red box). sgRNA expression can be driven by a U6 promoter or alternatively by a H1 promoter. The Cas9 component is currently available in three different forms: as a wild-type (hCas9) or a mutant (D10A) version for gene editing purposes and a catalytically dead (dCas9) version for gene silencing approaches. Exp-ression of Cas9 can be driven by any mammalian expression promoter (e.g., EF1A, CMV, etc.) or retroviral promoter (LTR). For nuclear targeting, the Cas9 gene requires multiple nuclear localization signals (NLS) and general expre-ssion of Cas9 can be enhanced by inclusion of a woodchuck post-transcriptional regulatory element (WPRE) at the 3′-end. A polyA-tail is required for expression vectors, while it should be deleted from lenti-/retroviral expression vectors that have a polyA recognition sequence in their 3′-LTR. Variations of this design are possible with respect to the Cas gene used or further modifications such as tagging the Cas gene with GFP or NLSs in order to optimize Cas9 nuclear targeting.