CRISPR/Cas9-Induced Knockout of Sting Increases Susceptibility of Zebrafish to Bacterial Infection

Abstract

Stimulator of interferon genes (STING) is an adapter protein that is activated when cyclic dinucleotides (CDNs) are present. CDNs originate from the cytosolic DNA of both pathogens and hosts. STING activation promotes efficient immune responses against viral infections; however, its impact in bacterial infections is unclear. In this study, we investigated the role of Sting in bacterial infections by successfully creating a sting-deficient (sting(−/−) with a 4-bp deletion) knockout zebrafish model using CRISPR/Cas9. The transcriptional modulation of genes downstream of cGAS (cyclic GMP-AMP synthase)-Sting pathway-related genes was analyzed in seven-day-old wild-type (WT) and sting(−/−) embryos, as well as in four-day-old LPS-stimulated embryos. The expression of downstream genes was higher in sting(−/−) than in healthy WT fish. The late response was observed in sting(−/−) larvae following LPS treatment, demonstrating the importance of Sting-induced immunity during bacterial infection by activating the cGAS–STING pathway. Furthermore, adult sting(−/−) fish had a high mortality rate and significantly downregulated cGAS–STING pathway-related genes during Edwardsiella piscicida (E. piscicida) infection. In addition, we assessed NF-κB pathway genes following E. piscicida infection. Our results show fluctuating patterns of interleukin-6 (il6) and tumor necrosis factor-α (tnfα) expression, which is likely due to the influence of other NF-κB pathway-related immune genes. In summary, this study demonstrates the important role of Sting against bacterial infection.

Suppressors of cGAS‐STING are downregulated during fin‐limb regeneration and aging in aquatic vertebrates

“…In fishes, the cGAS‐STING pathway has been shown to play an important role in their antiviral and antibacterial immune responses (Z. F. Liu et al, 2020; de Oliveira Mann et al, 2019; Sellaththurai et al, 2023). Our results extend these observations to the conservation of cGAS‐STING function in tissue regeneration and aging.…”

In Search of a Target Gene for a Desirable Phenotype in Aquaculture: Genome Editing of Cyprinidae and Salmonidae Species

“…The targets of studies include lysosomes [170,171], endoplasmic reticulum [172], membranes [173], membrane channels [174], cytoskeleton [175], recombination processes [176], aneuploidy [177], expression regulation [178], and signaling [179]. There is research on the genetic basis of the organism's response to oxidative stress [180][181][182][183][184], hypoxia [185][186][187], sodium ion uptake from the surrounding aquatic environment and ammonia excretion [188][189][190], inflammation processes [191,192], immunity [193][194][195][196] and disease resistance [197][198][199][200][201].…”

“…And knockout of them is not desirable. The sting [199] and tnf-α1 [341] genes are responsible for resistance to Edwardsiella piscicida, crp for resistance to Streptococcus pneumoniae [200], and cxc3.3 for resistance to Mycobacterium spp. [342].…”

A review on current advancement in zebrafish models to study chronic inflammatory diseases and their therapeutic targets

“…To overcome these challenges, accelerate the drug discovery and development processes, researchers around the globe are working on limiting these downfalls and developing target specific models for inflammation. CRISPR/Cas9 technology is widely integrated in zebrafish studies which can enable the researchers to generate zebrafish lines with precise gene mutations related to inflammation [ [180] , [181] , [182] ]. Moreover, for IBD, various studies are being performed to learn about the interaction between the zebrafish gut microbiome and the immune system [ 178 , 183 , 184 ].…”