Fig. 1

Fluorescent Phenotype Resulting from CRISPR Targeting of urod

(A) Schematic representation of urod coding sequence (CDS). The position of the CRISPR target sequences at the beginning of exons three and five are indicated.

(B) Outline of the experiment, Cas9 mRNA is injected into one-cell stage embryos along with a gRNA targeting either urod or p53 as a negative control (ctrl). Targeted mutation efficiency is assessed by T7E1 assay and sequencing, and the presence of red fluorescent erythrocytes by confocal microscopy.

(C) T7E1 mutagenesis assay at the CRISPR target site in the urod gene. The assay was performed on genomic DNA from 2 dpf embryos injected at the one-cell stage with Cas9 mRNA and either a gRNA against p53 (negative control) or a gRNA against urod. Cleavage bands (arrowheads) indicate the presence of mutations at the target site. See also Figure S1B.

(D) Confocal images reveal the presence of fluorescent blood cells in urod mosaic knockout embryos at 30 hpf. The black and white insets show a 2× magnification of the red fluorescent signal in the yolk region. See also Figure S1E.

(E) Most frequent (>1%) mutant urod alleles found by deep-sequencing in whole embryos injected with Cas9 mRNA and urod gRNA. The CRISPR target sequence is in green and mutations in red. The type of mutation and the associated frequency (in % of all mutated alleles) are indicated, large deletion (del).