Fig. 2.

Fig. 2.

On-target lamin B receptor (lbr) gene editing in neutrophils of Tg(mpx-cas9) zebrafish embryos. (A) Schematic of experimental steps for in vivo gene editing in neutrophils. (B) Zebrafish lbr locus showing gRNA target site in exon 2. (C) High-level on-target lbr gene editing from synthetic gRNA delivery. (Ci) Sanger sequencing chromatogram of wild-type (WT) whole-embryo DNA (upper panel; non-edited control) compared to that from F3 embryos injected with synthetic lbr gRNA complexed to exogenous Cas9 protein (middle panel; positive control) and neutrophil-lineage gene editing in fluorescence-activated cell sorting (FACS)-purified neutrophils from Tg(mpx-Cas9) embryos injected with synthetic lbr gRNA (lower panel). (Cii) Manhattan plot from next-generation sequencing (NGS) of the same neutrophil DNA preparation as Ci (lower panel), displaying cumulative distribution of aligned deleted alleles at the target locus. (Ciii) NGS of the same DNA preparation as Ci (lower panel) revealed six predominant variants (Var 1-6, bracketed Var 2 and 3 occurred in cis), representing 62.75% on-target gene editing. None of these variants was seen in DNA from embryos not injected with lbr gRNA. (Civ) Predicted amino acid sequences of variants 1-6. A high proportion of gene edits (70.28%), representing 44.1% of the NGS reads, are predicted to be nonsense mutations. (D) Low-level on-target lbr gene editing from plasmid gRNA delivery using a plasmid encoding lbr gRNA expressed from the U6 promoter (plasmid gRNA). (Di) Upper panel shows Sanger sequencing chromatogram of DNA from whole 3 dpf embryos injected with 1.5 ng/µl plasmid gRNA and exogenous Cas9 enzyme, serving as a positive control, showing low-level gene editing from plasmid gRNA delivery when Cas9 is in abundance. Lower panel shows Sanger sequencing chromatogram of DNA from FACS-purified neutrophils of 5 dpf Tg(mpx-Cas9) embryos injected with 30 ng/µl lbr plasmid gRNA alone, showing no detectable gene editing. (Dii) NGS of the same neutrophil DNA preparation as in Di (lower panel) detected one gene-edited variant (Var 1), representing 1.45% on-target gene editing. No gene editing was detected by NGS in ‘other cells’ from these same embryos. This variant was not seen in DNA from embryos not injected with lbr plasmid gRNA. (Diii) Predicted amino acid sequence of variant 1 results in a nonsense mutation. (E) No gene editing was detected by Sanger sequencing in FACS-purified macrophages from 3 dpf Tg(mpeg1:Cas9) embryos (right panel). FACS-purified neutrophils serve as an internal negative control (left panel). PAM, protospacer adjacent motif highlighted in red boxes and red font; red arrows indicate the sequencing direction; red asterisks mark sequence heterogeneity due to on-target gene editing; red dots indicate deletion; blue font, substituted nucleotides; green font, inserted nucleotides; purple font, truncated protein; black dots, sequence continues as WT.