Identification of homozygous F2 fish

For the generation of F2 individuals, the identified F1 fish were incrossed in order to obtain a statistical percentage of 25 % offspring that are homozygous for the mutant Pitx1 allele. At least eight F2 larvae were taken for each of the six lines in order to extract genomic DNA from them and subsequently carry out the PCR to amplify the Pitx1 locus. The PCR products were then subjected to Sanger sequencing in order to determine the respective mutant Pitx1 sequences.

Table 8. Pitx1 T19 F0 founder fishes and the indel mutations they inherit. Summary of all identified Pitx1 T19 founder fish. Sanger sequencing and alignment with the wild type Pitx1 sequence revealed the precise indel mutation in homozygous F2 larvae. On this basis, the resulting chances in the amino acid sequence were determined.

Founder Mutation Resulting changes in Pitx1 amino acid sequence

♀4 3 bp deletion Pro(12), Arg(13) are replaced with Phe

♂4 no mutation -

♂6 no mutation -

♂7 8 bp deletion Frameshift after Ser(8) and premature Stop codon after 48 aa

♂8 6 bp deletion Phe(9) and His(10) are missing

♂15 9 bp deletion Ser(8), Phe(9), His(10), Leu(11), Pro(12) are replaced with Met and Cys

In case of ♂4 and ♂6 F2 larvae only wild type Pitx1 sequences were obtained by the sequencing reaction. The sequencing profiles were consistently of good quality and, in addition, did not show obvious peak overlays indicating that there were no heterozygous larvae among them either (data not shown). This strongly supports the hypothesis derived from the TIDE assay that the founders ♂4 and ♂6 are actually false positives, even if the results of the T7E1 assay looked very promising.

In contrast to that, for all other lines founded by F0 fish ♀4, ♂7, ♂8 and ♂15, homozygous F2 larvae carrying a mutated Pitx1 allele were found. The mutations were all determined as deletions of a few base pairs (bp): 3 bp for ♀4, 8 bp for ♂7, 6 bp for ♂8 and 9 bp for ♂15 (Table 8). Thus the results are in conformity with the predictions obtained by TIDE (Fig. 42).

In case of the lines founded by ♀4, ♂7 and ♂15 the deletions were identified as three or multiples of three base pairs. A detailed examination of the mutant Pitx1 sequence revealed that, as expected, none of these lines contained a frameshift mutation, which would lead to a totally different amino acid sequence or even a premature Stop codon. Instead it was found that some amino acids were missing or have been replaced. The individual sequence

results for the Pitx1 locus in these three lines and the resulting amino acid sequences are visually presented in Fig. S26 and summarized in Table 8. Whether these changes in the Pitx1 protein sequence cause a phenotype in the larvae cannot be said with certainty yet. No obvious phenotype was found in F2 larvae from founder ♀4, ♂7 and ♂15 up to an age of 7 dpf. The exception were the F2 descendants of founder ♀4. Here, approximately 25 % of the F2 embryos exhibited a striking phenotype with enlarged and misshaped head and a pronounced pericardial edema at 48 hpf (Fig. S27). However, Sanger sequencing revealed that this phenotype is not connected to the mutation in the Pitx1 locus and thus might probably represent an off-target mutation in this line.

Pitx1 T19_♂7 F2

Fig. 43 Sequence information of homozygous Pitx1 T19 F2 fish. Genomic DNA was isolated from F2 larvae derived by founder fish ♂7. Sanger sequencing revealed the genotype of homozygous fish carrying the indel mutation of -8 base pairs. A total of ten base pairs were deleted and two new ones added, resulting in the overall deletion of eight base pairs. The consequence was a shift in the reading frame affecting the complete amino acid (aa) sequence after Ser(8) (framed in red). In addition a premature stop codon (*) was created after 48 aa. In combination this led to a truncated nonsense protein. Sanger sequencing carried out by eurofins.com. Alignment performed with SnapGene.

Establishment of a Pitx1 Knock-out line using CRISPR/Cas9 system

Sequencing Pitx1 locus of founder 7

The most promising indel mutation was found in the Pitx1 locus of homozygous F2 descendants from ♂7. The introduced deletion of eight bases causes a shift in the reading frame that results in a completely different amino acid sequence after Ser(8). Moreover, a premature Stop codon is created after 48 amino acids consequently resulting in a truncated protein (Fig. 43). Therefore the expectation was that this mutation causes a pronounced phenotype, which might even result in embryonic lethally. However, contrary to this hypothesis, larvae survived at least up to an age of 7 dpf with normal appearance and behaviour. WISH detecting Pitx1 transcripts revealed a normally developed pituitary gland at the 14-somite stage; examined in a complete clutch of the F2 generation (>50 embryos) (Fig. S28). Consequently, several F2 fish from founder ♂7 were raised to juveniles and adults in order to investigate a potentially reduction, malformation or lack of the pelvic fin. Each of 31 examined adult fish exhibited a normal sized pelvic fin (data not shown). Tail fin biopsy with subsequent genotyping revealed that 18 heterozygous (58 %) and 13 homozygous wild type fish (42 %) were among the 31 F2 individuals. The homozygous mutant Pitx1 allele was not detected in any adult F2 individual, although, statistically speaking, a number of 7-8 individuals would have been expected in 31 fish. This indicated, although the larva does not appear to have a phenotype, that the fish homozygously carrying the deletion of 8 bp in Pitx1 do not reach adulthood. The fitness of the larvae might be attributed to a partial compensation of the Pitx1 function by its close homologues Pitx2 and Pitx3, which are both expressed simultaneously with Pitx1 in the pituitary gland during early somitogenesis (Angotzi et al., 2008). Another prominent expression site of Pitx1 is the mandibular arch during early larval jaw development (Askary et al., 2017). A detailed examination of the correct organisation of the jawbones might therefore reveal a dysfunction of the mutated Pitx1 gene in the ♂7 F2 generation in future studies. Repeated examinations at regularly intervals between 7 dpf and 90 dpf should be performed to figure out the exact time frame and the cause at which the homozygous carriers die.