CRISPR nucleases are efficient tools to edit cellular genomes in a variety of organisms. However, the in vivo application of this technology is still severely limited by unwanted genomic cleavages, that are further increased by long-term expression of the nuclease and can lead to unpredictable results. To address this limitation, we developed a yeast-based assay which allows to simultaneously evaluate the on- and off-target activity towards two engineered genomic targets in order to select optimized Streptococcus pyogenes Cas9 (SpCas9) variants. The screening of SpCas9 variants obtained by random mutagenesis of the Rec1-II domain allowed the identification of hits with increased on/off ratios. Through the combination of the identified mutations within a single variant we isolated the best performing nuclease, that we named evoCas9 (evolved Cas9). Side by side analyses with recently reported rationally designed variants demonstrated a significant improvement in fidelity of our evoCas9. In addition, to control Cas9 persistence into cells over time, we developed a Self-Limiting Cas9 circuitry for Enhanced Safety (SLiCES) which consists of an expression unit for SpCas9, a self-targeting sgRNA and a second sgRNA targeting a chosen genomic locus. This self-limiting circuit, by controlling Cas9 levels, results in increased genome editing specificity. For its in vivo utilization, we 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 promoting its own neutralization through SpCas9 inactivation. The two strategies here developed represent complementary approaches to address a major issue in the genome editing field. On one hand, by preserving target cells from residual nuclease activity, our hit and go SLiCES system increases the safety margins for genome engineering. On the other, if compared to published structure-guided protein engineering approaches, our in vivo screening increases the likelihood to identify the best combination of amino acid substitutions for the generation of novel, error-free SpCas9 and could represent a valid strategy to enhance the specificity of other RNA-guided nucleases.
| Identifer | oai:union.ndltd.org:unitn.it/oai:iris.unitn.it:11572/368307 |
| Date | January 2017 |
| Creators | Casini, Antonio |
| Contributors | Casini, Antonio, Cereseto, Anna |
| Publisher | Università degli studi di Trento, place:TRENTO |
| Source Sets | Università di Trento |
| Language | English |
| Detected Language | English |
| Type | info:eu-repo/semantics/doctoralThesis |
| Rights | info:eu-repo/semantics/openAccess |
| Relation | firstpage:1, lastpage:137, numberofpages:137 |
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