Knockdown or Variants in exosc9 Cause Developmental Defects in Zebrafish
Zebrafish have previously been used as model systems for investigating variants in exosome complex subunits16, 17 and associated proteins19 and are consistently used for modeling the cerebellar, hindbrain, and motor neuron dysfunction observed in human disease. We concluded that zebrafish would therefore make a suitable in vivo disease model for the effects of reduced exosc9 function for investigating whether a phenotype consistent with the other exosomal models would result.
Injection of morpholino oligonucleotides and the CRISPR/Cas9 system were used to knock down or induce variants, respectively, in exosc9 in zebrafish embryos (Figure 5A). The exosc9 morpholino oligonucleotides led to aberrant splicing of the exosc9 transcript, which was confirmed via RT-PCR (Figure 5B), where morphant zebrafish had a retained intron that was confirmed by sequencing. In addition to the appearance of mis-spliced transcripts, the amount of wild-type (WT) exosc9 transcript was reduced in injected embryos. The embryos injected with Cas9 and gRNA for exosc9 would be expected to be mosaic; genomic DNA of cells would be a mixture of WT and various mutated forms of exosc9 in varying proportions. To confirm mutagenesis in the crispants, PCR with primers flanking the sgRNA target area was performed on genomic DNA. The PCR product was then cloned into the pGEM-T easy vector and colony PCR, and sequencing was performed on individual clones. Sequencing showed that there was a variation in the amount of mutagenesis occurring and that there was a phenotype-genotype correlation (Figure 5D).
Figure 5 Strategies Targeting exosc9 in Zebrafish
(A) Schematic of exosc9 in zebrafish demonstrating the sites to where the morpholino, gRNA, and primers were targeted.
(B) RT-PCR of zebrafish morphants. The morpholino caused the retention of an intron and a reduction of the WT product in a dose-dependent fashion. The identity of the bands was confirmed by Sanger sequencing.
(C) The target sequence for exosc9 gRNA and an example of a mutation found in a crispant.
(D) The mutation rate found in crispants of differing phenotypes.
Zebrafish injected with the morpholino oligonucleotide (morphants) and Cas9 and exosc9 sgRNA (crispants) developed a similar range of morphological phenotypes (Figure 6A). Mildly affected embryos had smaller heads and eyes, whereas severely affected embryos had extremely small, sometimes absent, eyes, very small heads, and truncated bodies (Figure 6A). The relative distribution of phenotypes was also similar in morphants and crispants (Figure 6B).
Figure 6 Knockdown of exosc9 in Zebrafish Causes Abnormal Morphology
(A) Representative morphological scoring of morphant and crispant exosc9 zebrafish embryos at 48 hpf. Normal, identical to uninjected control clutchmates; mild, smaller head and smaller eyes; severe, very small head, smaller or absent eyes, and misshapen body.
(B) Relative distribution of morphological phenotypes in exosc9 morphants and crispants at 48 hpf. Scale bar represents 1 mm.