Zebrafish Manipulations, CRISPR/Cas9 Editing, and Design of Morpholinos
Zebrafish animal experimentation was approved by the Ethical Committee for Animal Experimentation of the Geneva University Medical School and the Canton of Geneva Animal Experimentation Veterinary authority. Wild-type TU (Tübingen) zebrafish were maintained in standard conditions (26°C–28°C, water conductivity at 500 μS [pH 7.5]). Embryos obtained by natural matings were staged according to morphology/age.
Zebrafish kiaa1109 mutant lines were developed using CRISPR-Cas9-mediated genome editing. Using the ZiFiT online tool,19 we identified three suitable 20-nucleotide sites upstream of protospacer adjacent motifs (PAM) for S. pyogenes Cas9 and targeting kiaa1109 exons 1, 4, and 7 (numbering according to GenBank: NM_001145584.1). Annealed oligonucleotides carrying the 20-nucleotide target sequence were ligated into pDR274 (Addgene plasmid # 42250), and clones verified by Sanger sequencing, linearized, and used for in vitro transcription of single-guide RNAs (sgRNAs) using the MEGAshortscript T7 Transcription Kit (ThermoFisher). sgRNAs were mixed with recombinant Cas9 nuclease (PNA Bio), Danieau buffer, and phenol red as a tracer, and approximately 1 nL injected into early zebrafish embryos. Each injection contained 0.25 ng of sgRNA and 0.5 ng of Cas9 per nL. Evidence for genome editing was assessed qualitatively by PCR amplification around the target sites in each exon in injected embryo lysates. Heterogeneous PCR products, consistent with mosaic editing, was seen as smeared bands by gel electrophoresis, compared to uninjected embryos (not shown). Injected fish embryos were raised to adulthood and screened for their ability to transmit mutant kiaa1109 alleles by out-crossing and PCR genotyping. PCR products were cloned with pCRII TOPO (ThermoFisher) to separate alleles, and colony PCRs were sequenced to detect germline transmission of potential kiaa1109 frameshift alleles. Out-crossed F1 embryos were raised to adulthood for mutations detected in exon 1, 4, and 7, as separate lines. F1 adult fish were tail-clipped, targeted exons were amplified by PCR, and PCR products were cloned to pCRII TOPO (ThermoFisher) to identify specific kiaa1109 mutant alleles in heterozygosity by colony PCR and DNA sequencing. Heterozygous F1 fishes carrying the same kiaa1109 mutation were then in-crossed to assess embryonic survival and phenotype in homozygosity. kiaa1109 genotyping for embryos from these crosses was made using PCR, amplifying the target exon regions. Products from wild-type, heterozygous, or homozygous mutant amplicons were distinguished by gel electrophoresis. Details of the sgRNA target sites, representative mutant allele sequencing chromatograms, and predicted frameshifts for the three kiaa1109 mutant lines described are given in Figures S2 and S3.
To knock down kiaa1109 (GenBank: NM_001145584.1) in zebrafish, we designed two non-overlapping splice-blocking MOs (morpholinos) targeting pre-mRNA: (1) sbE4MO- 5′-TGTTCTGTTTTTGCACTGACCATGT-3′ and (2) sbE2MO- 5′-CAACATTGAGACAGACTCACCGATG-3′ (Gene Tools) that target the exon 4/intron 4 and exon 2/intron 2 boundaries, respectively. The standard Ctrl-MO (5′-CCTCTTACCTCAGTTACAATTTATA-3′) (Gene Tools) without any targets in the zebrafish genome was used for mock injections. MOs were dissolved in nuclease-free water and their concentrations determined by NanoDrop. The fish were injected at 1- to 2-cell stages (1–2 nL) using phenol red as a tracer in Danieau buffer. The following amounts of MO: 3.35 and 6.7 ng of sbE4MO; 5.6, 11.3, 16.9, and 22.3 ng of sbE2MO and the equivalent of Ctrl-MO for the higher doses were injected into wild-type zebrafish embryos, respectively. Uninjected, standard control MO, and kiaa1109 MO-injected embryos were collected at 2 dpf and total RNAs were isolated using standard Trizol protocol (Invitrogen). 1 μg of total RNA from each sample was used to synthesize cDNA with the Superscript III kit with Oligo d(T) primers (Invitrogen). Dilutions 1/20 of cDNA were used for standard PCR reactions (JumpStart RED Taq ReadyMix, Sigma-Aldrich). Basic quantifications of agarose gels were performed with ImageQuant TL software (GE Healthcare). We assessed embryos for morphological changes at 2 days post-fertilization. We grouped the embryos into four classes by morphology: normal embryos, embryos with clear midbrain and/or hindbrain ventricle swelling, curved embryos, and embryos with both phenotypes. The degree of hydrocephaly was not measured; hydrocephaly was assessed by clear deviation from the normal embryo morphology (see Results). Curved embryos showed caudal axis curvature. They were clearly distinguishable from the straight anterior-posterior axis of normal 2-day-old embryos (see Results). The most severely affected embryos had a combination of hydrocephaly and caudal axis curvature (see Results).