Plasmids and Oligonucleotides

The sgRNAs for the initial experiments on VSV-G vesicle production were transcribed from a U6 promoter-driven cassette derived from pX330 (a gift from Feng Zhang, Addgene 42230)1 and cloned into the pUC19 plasmid.13 sgRNA oligos were cloned using a double BbsI site inserted before the sgRNA constant portion according to a previously published cloning strategy.1 The Gag-SpCas9 plasmid was obtained through the fusion of the Gag coding sequence with 3×FLAG-SpCas9 encoded by the pX-SpCas9 vector.13 pX-SpCas9 was obtained by removal of an NdeI fragment including the sgRNA expression cassette from pX330 and was used to express SpCas9. MinimalGag-Cas9 was assembled in pCDNA3 by subcloning the SpCas9 coding sequence from pX-SpCas9 and MinimalGag from the Δ-Zwt-p2b plasmid,36 a generous gift from H.G. Gottlinger. Afterward, an additional version of both constructs was obtained by removing, through site-directed mutagenesis, a methionine in the linker peptide derived from pX-Cas9 that led to unfused SpCas9 production (Figure S4).

For VLP and VEsiCas production in BSR-T7/5 cells, sgRNAs were transcribed from a pVAX-T7-sgRNA expression plasmid having a T7 promoter-driven cassette cloned into the pVAX plasmid at the NdeI site. sgRNA oligos were cloned in pVAX-T7-sgRNA using a double BsmBI site inserted before the sgRNA constant portion (a list of oligonucleotides used to clone sgRNAs is available in Table S1). pVAX-T7-sgRNA included a 5′ HDV ribozyme33 designed to cleave the 3′ end of the sgRNA containing the T7 terminator and the ribozyme itself. The Cas9-nickase construct was obtained by inserting the D10A mutation in the SpCas9 coding sequence. The T7 RNA polymerase coding sequence was amplified from the genome of BSR-T7/5 cells and cloned in place of EGFP into the pEGFP-N1 plasmid using the HindIII/XbaI sites to obtain the pKANA-T7-RNA-Pol plasmid. Plasmids were verified by Sanger sequencing. Information regarding relevant plasmid DNA sequences produced for this manuscript can be found in the Supplemental Information.