Hit and go CAS9 delivered through a lentiviral based self-limiting circuit

In vivo application of the CRISPR/Cas9 technology is still limited by unwanted Cas9 genomic cleavages. Long term expression of Cas9 increases the number of genomic loci non-specifically cleaved by the nuclease. We developed a Self-Limiting Cas9 circuit for Enhanced Safety and specificity (SLiCES) which consists of an expression unit for Streptococcus pyogenes Cas9 (SpCas9), a self-targeting sgRNA and a second sgRNA targeting a chosen genomic locus. The self-limiting circuit by controlling Cas9 levels results in increased genome editing specificity. For its in vivo utilization, we next integrated SLiCES into a lentiviral delivery system (lentiSLiCES) via circuit inhibition to achieve viral particle production. Following its delivery into target cells, the lentiSLiCES circuit is switched on to edit the intended genomic locus while simultaneously stepping up its own neutralization through SpCas9 inactivation. By preserving target cells from residual nuclease activity, our hit and go system increases safety margins for genome editing.