Effects of CRISPR/Cas9-mediated dnd1 knockout impairs gonadal development in striped catfish.

Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology allows for the generation of loss-of-function mutations to enable efficient gene targeting to produce desired phenotypes, such as the production of germ cell-free fish. This technology could provide several applications for aquaculture and conservation of fisheries resources, such as the prevention of overpopulation in fish culture and gene flow from escaped farmed fish into wild populations and the production of germ cell-free recipient larvae for germ cell transplantation. This study aimed to develop CRISPR/Cas9 mediated dead-end 1 (dnd1) knockout techniques for striped catfish (Pangasianodon hypophthalmus). To optimise CRISPR/Cas9-induced dnd1 knockout, three single-guide RNAs (sgRNAs) were designed to target upstream sequences of start codon of the dnd1 gene. A combination of two concentrations of each sgRNA (100 and 200 ng/µl) and three concentrations of Cas9 (100, 250, and 500 ng/µl) was microinjected into fertilised striped catfish eggs. These sgRNAs/Cas9 could induce indel mutations and lower the primordial germ cell (PGC) numbers. Histological analyses indicated that sgRNA3 targeting upstream and nearest to the start codon at 200 ng/µL and Cas9 at 500 ng/µL showed the lowest PGC number. The reduction in PGC number was confirmed by in situ hybridisation using antisense dnd1 and vasa probes. All sgRNA/Cas9 combinations reduced the expression of dnd1, cxcr4b, dazl, nanos1, nanos2, and vasa, and the lowest expression levels were observed in gonads obtained from fish injected with 200 ng/µL sgRNA3 and 500 ng/µL Cas9 (P < 0.05). In addition, at 1 year of age, a significantly lower gonadosomatic index was observed in fish injected with all sgRNA and Cas9 at 500 ng/µL. Moreover, compared to the control fish, the ovaries and testes presented different morphologies in the sgRNA/Cas9-injected fish, that is, few previtellogenic oocytes in the ovary and spermatogonial cell-less testes. In conclusion, CRISPR/Cas 9 targeting dnd1 knockout at the upstream sequences of start codon was achieved, which resulted in the downregulation of dnd1 and lowered PGC number.

Comparison of Hyaluronic Acid Biosynthetic Genes From Different Strains of Pasteurella multocida.

Pasteurella multocida produces a capsule composed of different polysaccharides according to the capsular serotype (A, B, D, E, and F). Hyaluronic acid (HA) is a component of certain capsular types of this bacterium, especially capsular type A. Previously, 2 HA biosynthetic genes from a capsular type A strain were studied for the industrial-scale improvement of HA production. Molecular comparison of these genes across different capsular serotypes of P multocida has not been reported. This study aimed to compare 8 HA biosynthetic genes (pgi, pgm, galU, hyaC, glmS, glmM, glmU, and hyaD) of 22 P multocida strains (A:B:D:F = 6:6:6:4) with those of other organisms using sequence and structural bioinformatics analyses. These 8 genes showed a high level of within-species similarity (98%-99%) compared with other organisms. Only the last gene of 4 strains with capsular type F (HN07, PM70, HNF01, and HNF02) significantly differed from those of other strains (82%). Analysis of amino acid patterns together with phylogenetic results showed that the HA biosynthetic genes of the type A were closely related within the group. The genes in the capsular type F strain were notably similar to those of the capsular type A strain. Protein structural analysis supported structural similarities of the encoded enzymes between the strains of capsular types A, B, D, and F, except for the Pgm, GlmS, GlmU, and HyaD proteins. Our bioinformatics analytic workflow proposed that variations observed within these genes could be useful for genetic engineering-based improvement of hyaluronic acid-producing enzymes.