Fig. 4
Generation of transgenic mice using Nanoblades. a Left panel, scheme describing injection of mCherry VLPs or Nanoblades in the perivitelline space of mouse 1-cell embryos. Right panel, fluorescence microscopy of mouse blastocysts injected with mCherry VLPs at the single-cell stage. b Scheme of the design strategy to target the mouse Tyr locus (adapted from ref. 22). Upon editing and NHEJ repair, the HinfI restriction site becomes inactive. c Survival rates of injected embryos at two-cell, blastocyst, and newborn stage (the latter obtained from experiments presented in Supplementary figure 5). d T7 endonuclease (top panel) and HinfI restrictions (bottom panel) assays on PCR fragments amplified from the Tyr locus of Control or Nanoblades-injected embryos. e Top left panel, photographs of F0 mice generated from embryos injected with Nanoblades programmed with two sgRNAs targeting the Tyr locus. Top-right panel, phenotype, editing efficiency (as measured by TIDE analysis of the Sanger-sequencing electropherograms) and the main INDEL type as detected by Sanger sequencing of individual PCR clones. Bottom-panel, alignment of individual PCR clones obtained from the Tyr locus of F0 mice against the mouse mm10 genome indicating the main observed INDELs in chimeric mice (mouse #4, #7, and #8) and total excision of the Tyr sequence between the sgRNA1 and sgRNA2 targeting loci for the complete albino mouse (mouse #3). The Sanger sequencing electropherogram from the bulk PCR amplicon obtained from mouse #3 indicates complete editing at both targeted sites
