Extended Data Fig. 8: Somatic gene disruption efficiencies in zebrafish and recombinant Mmp25 catalytic domains production in E.coli.
From: A brain-specific angiogenic mechanism enabled by tip cell specialization
a,b, Targeting efficiencies (a) and SNPs and INDELs distributions (b) of embryos co-injected with 150 pg of zCas9 mRNA and 60 pg of the indicated targeting sgRNAs, as detected by Illumina amplicon deep sequencing. c, Coomassie blue staining of 400 ng of recombinant zebrafish and human his-tagged Mmp25 catalytic domains produced in E.coli, and purified on Ni-NTA resins. rzMmp25b: recombinant catalytic domain of zebrafish Mmp25, rhMMP25: recombinant catalytic domain of human MMP25. d, Western blot showing laminin-111 chains (black arrowheads) in 15 µg of Matrigel, after exposure or not to rhMMP25. e, Coomassie blue staining of 750 ng α1-antitrypsin (α1-AT, 2 µM), after exposure to 300 ng of rzMmp25b (2 µM) or 12 ng of rhMMP25 (75 nM). The black arrowheads point to α1-AT degradation products. The open arrowhead points to rzMmp25b. rhMMP25 is below detection limit. f,g, Quantification of hindbrain CtAs of control (mmp25a−/−;b+/−) and mmp25a/b−/− Tg(kdrl:EGFP) embryos at 36 hpf (f) and 48 hpf (g), injected or not (NI) at the one-cell stage with 150 pg of zCas9 mRNA and 60 pg of a serpina1 (sa1)-targeting sgRNA. Data represent median ± interquartile range. p-values were determined using the non-parametric two-tailed Mann-Whitney test. For f and g, n ≥ 10 embryos from 2 independent experiments.