One-step knock-in of two antimicrobial peptide transgenes at multiple loci of catfish by CRISPR/Cas9-mediated multiplex genome engineering.

CRISPR/Cas9-mediated multiplex genome editing (MGE) conventionally uses multiple single-guide RNAs (sgRNAs) for gene-targeted mutagenesis via the non-homologous end joining (NHEJ) pathway. MGE has been proven to be highly efficient for functional gene disruption/knockout (KO) at multiple loci in mammalian cells or organisms. However, in the absence of a DNA donor, this approach is limited to small indels without transgene integration. Here, we establish the linear double-stranded DNA (dsDNA) and double-cut plasmid (dcPlasmid) combination-assisted MGE in channel catfish (Ictalurus punctatus), allowing combinational deletion mutagenesis and transgene knock-in (KI) at multiple sites through NHEJ/homology-directed repair (HDR) pathway in parallel. In this study, we used single-sgRNA-based genome editing (ssGE) and multi-sgRNA-based MGE (msMGE) to replace the luteinizing hormone (lh) and melanocortin-4 receptor (mc4r) genes with the cathelicidin (As-Cath) transgene and the myostatin (two target sites: mstn1, mstn2) gene with the cecropin (Cec) transgene, respectively. A total of 9000 embryos were microinjected from three families, and 1004 live fingerlings were generated and analyzed. There was no significant difference in hatchability (all P > 0.05) and fry survival (all P > 0.05) between ssGE and msMGE. Compared to ssGE, CRISPR/Cas9-mediated msMGE assisted by the mixture of dsDNA and dcPlasmid donors yielded a higher knock-in (KI) efficiency of As-Cath (19.93 %, [59/296] vs. 12.96 %, [45/347]; P = 0.018) and Cec (22.97 %, [68/296] vs. 10.80 %, [39/361]; P = 0.003) transgenes, respectively. The msMGE strategy can be used to generate transgenic fish carrying two transgenes at multiple loci. In addition, double and quadruple mutant individuals can be produced with high efficiency (36.3 % âˆ¼ 71.1 %) in one-step microinjection. In conclusion, we demonstrated that the CRISPR/Cas9-mediated msMGE allows the one-step generation of simultaneous insertion of the As-Cath and Cec transgenes at four sites, and the simultaneous disruption of the lh, mc4r, mstn1 and mstn2 alleles. This msMGE system, aided by the mixture donors, promises to pioneer a new dimension in the drive and selection of multiple designated traits in other non-model organisms.

Mitochondrial DNA based diversity studies reveal distinct and substructured populations of pearlspot, Etroplus suratensis (Bloch, 1790) in Indian waters.

Pearlspot (Etroplus suratensis) is one of the most commercially important brackish water fish species widely found along the coastal regions of peninsular India and Sri Lanka. Pearlspot is known for its tender flesh, delectable taste, culinary tourism and highyielding market value. Information on the genetic makeup of stocks/populations is extremely vital as it forms the basis for future genetic studies. For this, we utilized ATPase6/8 genes of mtDNA of pearlspot populations collected from nine different locations ranging from Ratnagiri in Maharashtra state on the west coast to Chilika in Odisha on the east coast. Sequence analyses of these genes revealed 33 polymorphic sites, which include 17 singleton and 16 parsimony informative sites. Pair-wise genetic differentiation study (FST = 0.75) indicated significant (P<0.001) differences among all the pairs of stocks except those from Chilika and Nagayalanka. The spatial analysis of molecular variance (SAMOVA) significantly delineated the population into four groups (FCT = 0.69, P = 0.0001), namely northwest (Ratnagiri and Goa); southwest (Mangalore and lakes at Vembanad, Ashtamudi and Vellayani in Kerala); southeast (Pulicat in Tamil Nadu) and northeast (Chilika in Odisha and Nagayalanka in Andhra Pradesh). The above delineation is supported by clades of the phylogenetic tree and also the clusters of median joining haplotype network. The high haplotype diversity (0.84), low nucleotide diversity (0.003), and negative values of Tajima's D (-1.47) and Fu's Fs statistic (-14.89) are characteristic of populations having recently undergone demographic expansion. Mantel test revealed significant isolation by distance. The study identifies highly delineated structured populations with restricted gene flow. If such a stock is overfished, it is highly unlikely that it would recover through migration. For any future breeding programme in this species, it would be desirable to form a base population which incorporates the genetic material from all the locations so that we get a wide gene pool to select from.