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

Abstract

the application of genome engineering techniques to understand the mechanisms that regulate germ cell development opens promising new avenues to develop methods to control sexual maturation and mitigate associated detrimental effects in fish. In this study, the functional role of piwil2 in primordial germ cells (PGCs) was investigated in Nile tilapia using CRISPR/Cas9 and the resultant genotypes were further explored. piwil2 is a gonad-specific and maternally deposited gene in Nile tilapia eggs which is known to play a role in repression of transposon elements and is therefore thought to be important for maintaining germline cell fate. A functional domain of piwil2, PIWI domain, was targeted by injecting Cas9 mRNA and sgRNAs into Nile tilapia embryos at 1 cell stage. Results showed 54% of injected mutant larvae had no or less putative PGCs compared to control fish, suggesting an essential role of piwil2 in survival of PGCs. The genotypic features of the different phenotypic groups were explored by next generation sequencing (NGS) and other mutant screening methods including T7 endonuclease 1 (T7E1), CRISPR/Cas-derived RNA-guided engineered nuclease (RGEN), high resolution melt curve analysis (HRMA) and fragment analysis. Linking phenotypes to genotypes in F0 was hindered by the complex mosacism and wide indel spectrum revealed by NGS and fragment analysis. This study strongly suggests the functional importance of piwil2 in PGCs survival. Further studies should focus on reducing mosaicism when using CRISPR/Cas9 system to facilitate direct functional analysis in F0. Nile tilapia (Oreochromis niloticus) is one of the fastest growing farmed finfish species with > 120% increase in production volume over the last decade, such that global production has exceeded 4.1 million tonnes, worth USD 7.6 billion in 2017 1 making it the second largest (by volume) farmed finfish globally. While Nile tilapia is native to Northern Africa and Israel, it is now farmed widely out with its native range in many countries, contributing significantly to global food security particularly for poor rural communities 2. A significant hurdle that limited production potential of the species is precocious maturation where individuals direct energy towards sexual maturation to the detriment of somatic growth, which also results in the overproduction of unmarketable fry 3. This challenge has largely been overcome with the production of all male stocks which result in a more efficient production of tilapia with increased harvest weight 4. While this has had a significant beneficial impact on production of the species, contributing to its rapid expansion globally, the farming of reproductively competent animals has also resulted in widespread environmental impacts. Nile tilapia is considered to be an established invasive species in Asia 5 as well as Australia and North and South America, with reported impacts on native species and ecosystems 6. Therefore, there remains a need to develop methodologies to induce sterility and reduce production losses a...

Commensal bacteria‐immunity crosstalk shapes mucosal homeostasis in teleost fish

“…Moreover, CRISPR/ Cas techniques have been applied in several fish species, which will be favourable to detect efficient macrophage cytokines related to microbial colonization. 140,141 Dendritic cells (DCs) belong to the family of professional antigenpresenting cells (APCs) and are capable of directly uptaking both commensal and pathogenic bacteria in mammals. 142 In zebrafish and rainbow trout, DCs and its surface phenotypic molecules have been well identified.…”

Genome editing on finfish: Current status and implications for sustainability

“…Of the articles retrieved in this review, several reported mosaicisms in their research animals. 12,47,49,51,53,57,59,61,71 Straume et al 62 reported that mosaicism increased with higher…”

“…Kishimoto et al 13 found two mismatches for their small guiding RNAs (sgRNAs), however, Considering the discussion from the papers identified in this review, there is a further need to identify the presence of off-target and other unintended effects. This may imply to use recent developments as next-generation sequencing and multi-omics approaches, as seen approached in Jin et al 53 These methods need to be sensitive enough to distinguish between natural variation and mutations introduced by genome editing.…”

Genetic improvement technologies to support the sustainable growth of UK aquaculture

“…It has already been used experimentally to induce sterility in several aquaculture species. This can involve disrupting genes involved in sexual maturity, as seen in the Peruvian scallop, Argopecten purpuratus (Thresher et al 2014), or ablation of germ cells by knocking out the dnd gene in Atlantic salmon (Wargelius et al 2016;G€ uralp et al 2020) or piwil2 gene in Nile tilapia (Jin et al 2020). These demonstrations of gene editing technology to control sterility open the door to targeting many other desirable traits.…”