(a) Each triangle represents an individual animal injected with 16.5/1,200 pg tjp1b_L sgRNA/Cas9-encoding mRNA. Spinal cords were analyzed for the loss of synapses and heads were assayed for mutational efficiency on the basis of qPCR analysis of the tjp1b locus. We found that heads and spinal cords had indistinguishable mutational efficiencies (heads, 0.59 ± 0.04; tail, 0.61 ± 0.05; P = 0.84; values are average ± s.e.m. compared by Student’s t-test). The square of the mutational efficiency is plotted on the x-axis on the basis of the expectation that phenotypes are expected to be due to biallelic loss of a gene. There is a strong correlation of genotype to phenotype (R2 = 0.98). The lines represent the expected genotype-phenotype association, assuming random mutagenesis and that deleterious alleles occur only with out-of-frame mutations (dotted line, y = (2/3)2x) or both in-frame and out-of-frame mutations (solid line, y = (3/3)2x). The fact that the phenotypic rate approaches 100% suggests either that in-frame deletions are deleterious or that heterozygous loss of tjp1b effects synapse formation. Note that we found that the qPCR genotypic analysis underestimated the mutational efficiency by 6%–15% (see Online Methods). This underestimation would result in an increased slope of genotype-phenotype association plotted here. (b–d) Images are 15-µm dorsal-view projections of two spinal cord segments at 5 dpf. Anterior is to the left. Scale bar, 10 µm. Larvae were stained for Connexin36 (Cx36, white) to mark the electrical synapses and for neurofilaments (RMO44, red) to mark neuronal processes, including M and CoLo. Individual Cx36 channel is shown in neighboring panel. Heterozygous loss of tjp1b has no effect on M synapse formation, whether the germline-transmitted mutation is out-of-frame (b, tjp1bfh449/+) or in-frame (c, tjp1bfh451/+). However, trans-heterozygous tjp1b mutants carrying an out-of-frame and an in-frame mutation (tjp1bfh449/fh451) lose electrical synapses, confirming that in-frame mutations are deleterious at this locus (d). In a, N = 32 embryos.