Development of an intein-mediated split–Cas9 system for gene therapy

Abstract

Using CRISPR/Cas9, it is possible to target virtually any gene in any organism. A major limitation to its application in gene therapy is the size of Cas9 (>4 kb), impeding its efficient delivery via recombinant adeno-associated virus (rAAV). Therefore, we developed a split–Cas9 system, bypassing the packaging limit using split-inteins. Each Cas9 half was fused to the corresponding split-intein moiety and, only upon co-expression, the intein-mediated trans-splicing occurs and the full Cas9 protein is reconstituted. We demonstrated that the nuclease activity of our split-intein system is comparable to wild-type Cas9, shown by a genome-integrated surrogate reporter and by targeting three different endogenous genes. An analogously designed split-Cas9D10A nickase version showed similar activity as Cas9D10A. Moreover, we showed that the double nick strategy increased the homologous directed recombination (HDR). In addition, we explored the possibility of delivering the repair template accommodated on the same dual-plasmid system, by transient transfection, showing an efficient HDR. Most importantly, we revealed for the first time that intein-mediated split–Cas9 can be packaged, delivered and its nuclease activity reconstituted efficiently, in cells via rAAV.

CRISPR-Based Technologies for the Manipulation of Eukaryotic Genomes

“…These variants, including an intein-inactivated Cas9 system (Davis et al, 2015) and a small molecule-dimerized split Cas9 system (Zetsche et al, 2015b), have been shown to substantially improve genome editing specificity in mammalian cells compared with wild-type Cas9 by carefully controlling the temporal window within which active Cas9 is generated so that less active Cas9 is present after modification of the on-target loci is complete (Figure 2g,h). Similar systems, such as light-activated Cas9 variants (Nihongaki et al, 2015a;Hemphill et al, 2015;Jain et al, 2016), split Cas9 variants (Truong et al, 2015;Wright et al, 2015), small-molecule induction of Cas9 (Dow et al, 2015), and an engineered allosterically regulated Cas9 (Oakes et al, 2016) could also be used to reduce off-target genome editing following these same principles.…”

dCas9: A Versatile Tool for Epigenome Editing

“…An alternative strategy to control the binding of Cas9 to its target sequence relies on intein-mediated splicing (Davis et al, 2015;Truong et al, 2015). Designs with intein in the reading frame of fulllength Cas9 or fused to both fragments of split-Cas9 have been used.…”

“…Designs with intein in the reading frame of fulllength Cas9 or fused to both fragments of split-Cas9 have been used. In both cases, after intein transsplicing is induced full-length functional Cas9 is obtained (Davis et al, 2015;Truong et al, 2015). An other, less common approach, is to use Cas9 inactive due to caging of lysine residues necessary for Cas9 function (Hemphill et al, 2015).…”

“…The use of such small orthologues reduces the size problem with no drawbacks on activity, as observed for a truncated SpyCas9 derivative in which a less-conserved, non-interacting portion of the recognition lobe had been removed (Nishimasu et al, 2014). The split-dCas9 system also provides a solution to the size problem, but it requires two vectors for delivery (Truong et al, 2015). Alternatively, cells stably expressing dCas9-effector proteins can be complemented by delivery of sgRNA, but stable expression is difficult to achieve when using primary cells or multicellular organisms.…”

Gene Editing for Treatment of Neurological Infections

“…However, AAV is limited by the small size of the transgene that can be accommodated. There are a number of approaches to this problem, including delivering Cas9 and gRNAs in separate AAV vectors [68], splitting the Cas9 enzyme into halves that are delivered separately [69], or using a split-Cas9 with split-inteins and intein-mediated trans-splicing reconstitution of the full-length Cas9 protein [70]. Ran et al [71] reported that Cas9 from Staphylococcus aureus (SaCas9) is able to edit with similar efficiency to Cas9 from Streptococcus pyogenes but is > 1 kb shorter.…”