Generation of biallelic F0 mutants in medaka using the CRISPR/Cas9 system

Abstract

Several animal models generated by genome editing methods develop somatic mosaic mutations including wild-type genome sequence in F0 generation because it is difficult to use editing tools at the one-cell stage. Producing complete knockout animals quickly is a great advantage in determining the function of target genes. This study investigated the generation of F0 knockout medaka using the CRISPR/Cas9 system. To determine whether this editing system induced mutations in the medaka genome at the one-cell stage, recombinant Cas9 protein, tracrRNA and crRNA for dead end (dnd), which is essential for germ cell development, were injected into one-cell stage embryos of olvas-DsRedExpress transgenic medaka. This allowed germ cells to be visualized by DsRed fluorescence. Genomic DNA extracted from embryos at the one-cell stage was analyzed by sequencing. Predictably, biallelic mutated sequence patterns in the target sites of dnd were found in the injected embryos. To investigate the phenotypes of the mutated fish, fluorescent and histological observations of germ cells were carried out using fry and adults. The mutations resulted in a complete loss of germ cells, suggesting loss of function of dnd in the injected embryos. Therefore, this system appears to be extremely effective for the production of F0 knockout medaka.

Transcriptional Regulation of Müllerian Inhibiting Substance (MIS) and Establishment of a Gonadal Somatic Cell Line Using mis-GFP Transgenic Medaka (Oryzias latipes)

“…To investigate whether the immortalized hybridomas express the gonadal somatic cell markers, we examined germ cell and gonadal somatic cell markers using the hybridomas cultured for 3 weeks. qRT-PCR showed that the germ cell marker dead end (dnd) ( 32 ) was not expressed in any of the cell lines examined in this study ( Figure 4E ). Conversely, the Sertoli cell marker mis was expressed weakly in FOT-01 and FOT-02, and strongly in OLHE-131 ( Figure 4F ).…”

“…A small laboratory fish with an XX/XY sex determination system, it has advantages such as a short generation time, small genome size, and several useful strains are available ( 29 ). Additionally, transgenesis, knockdown techniques, and genome editing using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 have been established ( 30 – 32 ). Medaka is therefore a valuable vertebrate model for the analysis of the molecular genetics of various biological phenomena, including embryonic development and sex differentiation.…”

Decode-seq: a practical approach to improve differential gene expression analysis

“…In fact, Decode-seq also identified the germ cell marker vasa as a DEG in the top 300 list. Lastly, we knocked out a novel DEG (ENSORLG00000007290) using a rapid knockout method to generate F0 mutants [27,28]. The mutant showed severe germ cell depletion ( Fig.…”

Physiological impact and comparison of mutant screening methods in piwil2 KO founder Nile tilapia produced by CRISPR/Cas9 system

“…One of the challenges of applying CRISPR/Cas9 methodologies in poikilothermic species like fish is that temperature conditions in vivo will be suboptimal for Streptococcus pyogenes Cas9 (SpCas9) activity which is optimal at 37 °C 19 . Although editing activity with the CRISPR/Cas9 has been reported in tropical species like zebrafish, medaka and Nile tilapia at 26-28 °C 13,15,20 , the apparent diversity in resultant individual genotypes requires careful analysis and interpretation. To date, CRISPR/Cas9-mediated sterility studies have lacked a comprehensive screening and understanding of genotypes generated in F0 animals due to biased mutant screening methods, lack of standardisation and/or methodological details.…”

“…The lack of understanding of the resultant mutations including the level of mosaicism and the indel spectrum hinders the direct functional analysis in the injected animals. The most frequently used screening methods in gonad-related gene functional studies have been restriction enzyme digestion (RED) and Sanger sequencing of a limited number of cloned sequences [8][9][10][11][12][13]15,[21][22][23] , with a few studies adopting high resolution melt curve analysis (HRMA) or SURVEYOR techniques 24,25 . Such approaches have a number of potential limitations in their accuracy to describe KO effects on target genotypes, which are further confounded by the pooling of samples which ultimately provides a biased interpretation of the efficacy of CRISPR/Cas9 gene editing in this field.…”