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

Gianluca Petris (), Antonio Casini, Claudia Montagna, Francesca Lorenzin, Davide Prandi, Alessandro Romanel, Jacopo Zasso, Luciano Conti, Francesca Demichelis and Anna Cereseto ()
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Gianluca Petris: Centre for Integrative Biology (CIBIO), University of Trento, Laboratory of Molecular Virology
Antonio Casini: Centre for Integrative Biology (CIBIO), University of Trento, Laboratory of Molecular Virology
Claudia Montagna: Centre for Integrative Biology (CIBIO), University of Trento, Laboratory of Molecular Virology
Francesca Lorenzin: Centre for Integrative Biology (CIBIO), University of Trento, Laboratory of Computational Oncology
Davide Prandi: Centre for Integrative Biology (CIBIO), University of Trento, Laboratory of Computational Oncology
Alessandro Romanel: Centre for Integrative Biology (CIBIO), University of Trento, Laboratory of Computational Oncology
Jacopo Zasso: Centre for Integrative Biology (CIBIO), Laboratory of Stem Cell Biology
Luciano Conti: Centre for Integrative Biology (CIBIO), Laboratory of Stem Cell Biology
Francesca Demichelis: Centre for Integrative Biology (CIBIO), University of Trento, Laboratory of Computational Oncology
Anna Cereseto: Centre for Integrative Biology (CIBIO), University of Trento, Laboratory of Molecular Virology

Nature Communications, 2017, vol. 8, issue 1, 1-9

Abstract: Abstract 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. Here we develop 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 results in increased genome editing specificity by controlling Cas9 levels. For its in vivo utilization, we next integrate SLiCES into a lentiviral delivery system (lentiSLiCES) via circuit inhibition to achieve viral particle production. Upon delivery into target cells, the lentiSLiCES circuit switches 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.

Date: 2017
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DOI: 10.1038/ncomms15334

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