CRISPR StAR: sgRNA expression vectors for reliable and reproducible in vivo screenings

Technology Description

CRISPR StAR is based on CRISPR switch (see Partnering Opportunity for 44-00004), and takes advantage of sgRNA expression vectors, in which the sgRNA gene is interrupted by a disruption cassette flanked by a pair of recombinase recognition sites, which upon recombination facilitate the formation of an active sgRNA. However, the CRISPR StAR vector also carries a second pair of recombinase recognition sites which upon recombination facilitates the formation of an inactive sgRNA that serves as an internal control. Both recombination events are mutually exclusive and strictly stochastic, and accordingly yield a specific ratio of active and inactive sgRNA. In negative selection screens, the innovative sgRNA constructs are activated only after their representation has recovered. True essential genes are determined not only by depletion of the corresponding sgRNA (which is per se difficult to distinguish from a lack of transduction or recovery), but rather by a stochastic drift in the ratio of active and inactive sgRNA.

The figure shows the Volcano blots of a CRISPR screen in intestinal organoids using either state-of-the-art methodology (left) or CRISPR StAR (right). Each dot in these graphs represents a gene targeted by the sgRNA library employed. In an ideal world, the dots should center around the middle, and essential genes should be called with higher p-values (y-axis). As shown, only with CRISPR StAR, the dots are nicely centered, and top hits are identified with high confidence in the graph’s upper part. Furthermore, and in contrast to the conventional method, the identification of essential genes is highly reproducible, as indicated by the colored dots. In summary, CRISPR StAR enables the detection of essential genes with high confidence even in heterogenous system.