FIGURE 4

FIGURE 4

Generation of coilin N‐terminal (ΔN) mutants in zebrafish using CRISPR/Cas9. (A) Schematic of coilin's DNA and amino acid sequences showing the typical translational start site (black arrow), the PAM and sgRNA target sequences used to engineer CRISPR/Cas9 mutagenesis (red and green bars), and the alternative translation start (purple double bar) site lacking a Kozak sequence (red dash). The location of a SNP (A/T) corresponding to the N base of the NGG PAM sequence is shown. (B) Illustration of the development of homozygous COIL ΔN mutants. (C) Chromatogram illustrating DNA sequencing results which display the four base pair deletion characterizing the ΔN1 mutation (forward direction) and the eight‐base pair deletion charactering the ΔN2 mutation (reverse direction). The 4 base pair deletion strain has a T at the SNP position and the 8 base pair deletion stain has an A (T when using reverse primer) at this position. Red dash boxes are indicating the deleted sequences and lighting bolts are indicating the site of double‐strand break. (D) Histogram displaying the qPCR results of coilin mRNA expression in COIL ΔN1 and ΔN2 fish relative to wild‐type (WT) zebrafish. Data represents 3 biological replicates with 2 technical repeats for a total N = 6. Error bars represent SD and black dots represent individual data points. ****p < 0.0001 for comparisons to WT. (E) Western blot visualizing coilin protein expression in the testis of COIL ΔN1 and ΔN2 fish relative to WT zebrafish and quantification (histogram). ***p < 0.001.