Fig. 3

“All-in-one” Nanoblades for knock-in experiments and assessment of Nanoblades off-target activity. a Left panel, Nanoblades targeting human DDX3 close to its start codon were complexed with a donor ssDNA bearing homology arms to the targeted locus and a Flag-tag sequence in the presence of polybrene. HEK293T cells were then transduced with these “All-in-one” Nanoblades. After cell amplification, a fraction of cells were collected to extract genomic DNA and total proteins while the remaining cells were cultured to obtain single-cell clonal populations. Right panel, insertion of the Flag-tag in HEK-293T cells transduced with “all-in-one” Nanoblades complexed with increasing amounts of donor ssDNA was assessed by Flag-immunoprecipitation followed by western-blot using anti-flag or anti-DDX3 antibodies in the input and Flag-immunoprecipitation elution fractions. Flag insertion was also assessed by PCR using a forward primer in the flag-sequence and a reverse primer in the DDX3 locus (Orientation PCR assay) or using primers flanking the Flag sequence (Insertion PCR assay). Bottom panel, Flag-insertion in 20 different single-cell-derived clones was assessed by PCR using primers flanking the Flag-sequence. b Left panel, off-target monitoring in immortalized mouse macrophages stably expressing GFP transgenes bearing silent mutations in the region targeted by the sgRNA. Right panel, cells were transfected with plasmids coding for Cas9 and the sgRNA or transduced with Nanoblades. GFP expression was measured by FACS 72 h after transfection/transduction (n = 3). c Left and right panels, gene-editing at the EMX1 on-target site and the MFAP1 intronic off-target site measured by high-throughput sequencing in untreated cells (control cells) and cells transduced with EMX1 Nanoblades (Nanoblades) or transfected with plasmids coding for Cas9 and the EMX1 sgRNA (DNA transfection) (n = 3). Statistical significance of the Nanoblades and DNA transfection comparison at the on-target site was computed using a two-tail Student test. d Left panel, position of sgRNAs targeting the promoter of TTN and VLPs with different combination of sgRNAs produced for the experiment. Right-panel, TTN mRNA expression levels (normalized to Control) as measured by qPCR in MCF7 transduced with VLPs (n = 3). Error bars in all figures correspond to standard deviation