Experiments in mice show that designed epigenome editors that contain domains of transcriptional repressors can enable stable epigenetic silencing of Pcsk9, a gene with a role in cholesterol homeostasis, without inducing DNA breaks.
Molecular cloning and mRNA production ETRs were transiently delivered as either plasmid DNA (in cell lines) or mRNA (in cell lines, primary cells and in vivo). To this end, ETRs were cloned in an expression vector containing: (i) an upstream CMV promoter; (ii) an upstream T7 promoter for mRNA in vitro transcription (IVT); (iii) a downstream WPRE signal; (iv) a 3′-terminal stretch of 64 adenines (64A); and (v) a SpeI plasmid linearization site for mRNA IVT (Extended Data Fig. 1b). The coding sequences of the dCas9-based ETRs were previously described15. For plasmid-mediated gRNA expression, the crRNA sequences were cloned into a previously described expression vector containing a U6 promoter and the sequence of the Staphylococcus pyogenes Cas9 trRNA39. gRNAs targeting the CGI of the mouse Pcsk9 were designed using Chop Chop40 (https://chopchop.cbu.uib.no/) and selected according to high simulated activity and specificity. ZFPs and TALEs were designed and synthesized by Merck and Thermo Fisher Scientific, respectively, and subcloned into the mammalian expression plasmid in place of the dCas9 sequence. mRNAs were produced by IVT using the T7 Megascript Kit (Thermo Fisher Scientific, AMB1334-5) according to the manufacturer's instructions. For the in vitro experiments, partially modified mRNAs were produced by IVT, including the following modifications to the standard protocol: (i) inclusion of the anti-reverse cap analogue 3´-O-Me-m7G(5′)ppp(5′)G (NEB, M0251) at a final concentration of 8 mM; and (ii) reduction of the GTP concentration from 7.5 to 2.5 mM. For the in vivo experiments, heavily modified mRNAs were produced by IVT, including the following modifications to the standard protocol: (i) inclusion of CleanCap-AG (Trilink BioTechnologies, N-7113) at a final concentration of 4 mM; and (ii) substitution of UTP with N1-Met-ψ-Uridine (Trilink BioTechnologies, N-1081) at a final concentration of 7.5 mM. mRNAs were then purified using the RNeasy Mini Kit (Qiagen,…
Cappelluti, Martino Alfredo, San Raffaele Telethon Institute For Gene Therapy, Irccs San Raffaele Scientific Institute, Milan, Mollica Poeta, Valsoni, Quarato, Merlin, Department Of Health Sciences