Anna Cereseto

Wednesday, 24 October 2018 | 2:00 pm

Centre for Integrative Biology (CIBIO)University of Trento, ITALY

From user- to genome-friendlyCRISPR/Cas9

(Host: M. Giacca)

Therapeutic applications of Cas9 are still limited by efficientin vivo delivery and unspecific cleavages. In the attemptto tackle these issues, we have generated more specificSpCas9 variants and a traceless-viral based delivery system.The identification of high-specificity SpCas9 variants from arandom library was obtained through a yeast-based assayallowing simultaneous evaluation of on- and off-targetactivity.

The combination of the best isolated substitutionstogether with structure-assisted prediction generated anevolved Cas9 (evoCas9), characterized by unprecedented fidelity. Since viral-based delivery of Cas9 is associated withnon-specific cleavages due to its long-term expression,we developed a Self-Limiting Cas9 circuitry deliveredthrough a lentiviral vector for Enhanced Safety andspecificity (lentiSLiCES).

Upon delivery into target cells,the lentiSLiCES circuit is switched on to edit the intendedgenomic locus while simultaneously stepping up its ownSpCas9 inactivation. The strategies developed in our laboratory represent astep forward towards safer and efficient genome editing.