Kiran Dashrath Rasal

Establishing targeted carp TLR22 gene disruption via homologous recombination using CRISPR/Cas9.

Recent advances in gene editing techniques have not been exploited in farmed fishes. We established a gene targeting technique, using the CRISPR/Cas9 system in Labeo rohita, a farmed carp (known as rohu). We demonstrated that donor DNA was integrated via homologous recombination (HR) at the site of targeted double-stranded nicks created by CRISPR/Cas9 nuclease. This resulted in the successful disruption of rohu Toll-like receptor 22 (TLR22) gene, involved in innate immunity and exclusively present in teleost fishes and amphibians. The null mutant, thus, generated lacked TLR22 mRNA expression. Altogether, this is the first evidence that the CRISPR/Cas9 system is a highly efficient tool for targeted gene disruption via HR in teleosts for generating model large-bodied farmed fishes.

Analysis of consequences of non-synonymous SNP in feed conversion ratio associated TGF-β receptor type 3 gene in chicken

The recent advances in high throughput sequencing technology accelerate possible ways for the study of genome wide variation in several organisms and associated consequences. In the present study, mutations in TGFBR3 showing significant association with FCR trait in chicken during exome sequencing were further analyzed. Out of four SNPs, one nsSNP p.Val451Leu was found in the coding region of TGFBR3. In silico tools such as SnpSift and PANTHER predicted it as deleterious (0.04) and to be tolerated, respectively, while I-Mutant revealed that protein stability decreased. The TGFBR3 I-TASSER model has a C-score of 0.85, which was validated using PROCHECK. Based on MD simulation, mutant protein structure deviated from native with RMSD 0.08 Å due to change in the H-bonding distances of mutant residue. The docking of TGFBR3 with interacting TGFBR2 inferred that mutant required more global energy. Therefore, the present study will provide useful information about functional SNPs that have an impact on FCR traits.

Simple sequence repeats (SSRs) markers in fish genomic research and their acceleration via next-generation sequencing and computational approaches

AbstractSimple sequence repeats (SSRs) are becoming a choice of markers in fish genetic research due to their abundance in the genome, co-dominant nature, high polymorphism and ability to reproduce. Thus, in this review, we have discussed regarding SSRs markers developed in fishes using different techniques. These markers have been used for revealing genetic variability, strain and species identification, genetic linkage map construction and parentage assignment in fish genetic research. Recently, high-throughput sequencing platform has been widely used in non-model fishes for genome/transcriptome sequencing to understand genomic information. The rapid progress in fish genomic research has been made due to sequencing platform along with their low cost for sequencing and use of the advanced computational tools for generated data analysis. We have shown that different next-generation sequencing platforms have been applied in the genomic studies for SSRs markers identification in fishes with evidence. We have depicted the use of various computational tools/algorithms for the SSRs identification from genome/transcriptome data. However, we also highlighted existing challenges in high-throughput sequencing data analysis as well as the current need of computationally deep analysis software/tools/expertise. The purpose of this review is to get envisage on the various possibilities, which can be harnessed via these new technologies and advanced computational tools for SSRs marker development via genome/transcriptome sequencing of aquaculture species.

Gene editing tools: state-of-the-art and the road ahead for the model and non-model fishes

Advancements in the DNA sequencing technologies and computational biology have revolutionized genome/transcriptome sequencing of non-model fishes at an affordable cost. This has led to a paradigm shift with regard to our heightened understandings of structure-functional relationships of genes at a global level, from model animals/fishes to non-model large animals/fishes. Whole genome/transcriptome sequencing technologies were supplemented with the series of discoveries in gene editing tools, which are being used to modify genes at pre-determined positions using programmable nucleases to explore their respective in vivo functions. For a long time, targeted gene disruption experiments were mostly restricted to embryonic stem cells, advances in gene editing technologies such as zinc finger nuclease, transcriptional activator-like effector nucleases and CRISPR (clustered regulatory interspaced short palindromic repeats)/CRISPR-associated nucleases have facilitated targeted genetic modifications beyond stem cells to a wide range of somatic cell lines across species from laboratory animals to farmed animals/fishes. In this review, we discuss use of different gene editing tools and the strategic implications in fish species for basic and applied biology research.

MicroRNA in aquaculture fishes: a way forward with high-throughput sequencing and a computational approach

Current progress in high-throughput sequencing has opened up avenues to produce massive quantities of sequencing data from non-model fishes at an affordable cost. Thus, data analysis is also evolving at a rapid pace because of cutting edge computational tools. With the development and availability of experimental technologies and computational approaches, the field of MicroRNA (miRNA) biology has advanced over the last decade. MicroRNAs can play an important role in gene modulation via post-transcriptional gene regulation during acclimation and adaptation, in case of adverse conditions or climate change for example. These are useful and substantial contributors to regulatory networks of development and adaptive plasticity in fishes. Next generation sequencing technologies have extensively been used for solving biological questions in non-model fishes, where data pertaining to genome or transcriptome are either scant or totally unavailable. The data generated through this process have been used for gene discovery, variant identification, marker discovery and miRNA identification. Here, we discuss the role of miRNA in gene regulation pertaining to fish and its investigation via sequencing platforms, as well as the current use of computational algorithms for miRNA analysis. The purpose of this review is to examine the use of miRNA in aquaculture and further to investigate new technologies and advanced computational tools. However, our review also emphasizes existing challenges for miRNA investigations carried out via high-throughput sequencing and the growing demand for computationally intensive analysis software. This work along with assembled information on the known miRNAs in fish species will be useful while undertaking future studies for understanding the role of miRNAs.

Identification of Deleterious Mutations in Myostatin Gene of Rohu Carp (Labeo rohita) Using Modeling and Molecular Dynamic Simulation Approaches

The myostatin (MSTN) is a known negative growth regulator of skeletal muscle. The mutated myostatin showed a double-muscular phenotype having a positive significance for the farmed animals. Consequently, adequate information is not available in the teleosts, including farmed rohu carp, Labeo rohita. In the absence of experimental evidence, computational algorithms were utilized in predicting the impact of point mutation of rohu myostatin, especially its structural and functional relationships. The four mutations were generated at different positions (p.D76A, p.Q204P, p.C312Y, and p.D313A) of MSTN protein of rohu. The impacts of each mutant were analyzed using SIFT, I-Mutant 2.0, PANTHER, and PROVEAN, wherein two substitutions (p.D76A and p.Q204P) were predicted as deleterious. The comparative structural analysis of each mutant protein with the native was explored using 3D modeling as well as molecular-dynamic simulation techniques. The simulation showed altered dynamic behaviors concerning RMSD and RMSF, for either p.D76A or p.Q204P substitution, when compared with the native counterpart. Interestingly, incorporated two mutations imposed a significant negative impact on protein structure and stability. The present study provided the first-hand information in identifying possible amino acids, where mutations could be incorporated into MSTN gene of rohu carp including other carps for undertaking further in vivo studies.

In Silico Analysis of nsSNPs of Carp TLR22 Gene Affecting its Binding Ability with Poly I:C

Immune response mediated by toll-like receptor 22 (TLR22), only found in teleost/amphibians, is triggered by double-stranded RNA binding to its LRR (leucine-rich repeats) ecto-domain. Accumulated evidences suggested that missense mutations in TLR genes affect its function. However, information on mutation linked pathogen recognition for TLR22 was lacking. The present study was commenced for predicting the effect of non-synonymous single-nucleotide polymorphisms (nsSNPs) on the pathogen recognizable LRR domain of TLR22 of farmed carp, Labeo rohita. The sequence-based algorithms (SIFT, PROVEAN and I-Mutant2.0) indicated that three SNPs (out of 27) such as p.L159F (rs76759876) and p.L529P (rs749355507) of LRR, and p.I836M (rs750758397) of intracellular motifs could potentially disrupt protein function. The 3D structure was generated using MODELLER 9.13 and further validated by SAVEs server. The simulated molecular docking of native TLR22 and mutants with poly I:C ligand indicated that mutations positioned at p.L159F and p.L529P of the LRR region affects the binding affinity significantly. This is the first kind of study of predicting nsSNPs of teleost TLR22 with disturbed ligand binding affinity with its extra-cellular LRR domain and thereby likely hindrance in subsequent signal transduction. This study serves as a guide for in vivo evaluation of impact of mutation on immune response mediated by teleost TLR22 gene.