**7. Conclusion and remarks**

sequencing technologies has increased throughput studies of ncRNAs considerably. Consequently, studies on ncRNAs have contributed toward better understanding of disease resistance, productivity, breeding and meat quality in livestock species [203]. Although the numbers of detected ncRNA transcripts are increasing continuously, the ncRNAs identified and annotated in livestock species are still very scanty, compared with human data. Therefore, there is need to continue to explore the ncRNA transcriptome of livestock species [204]. The ability to explore and modify the genomes of livestock species could be beneficial in improving disease resistance, productivity, breeding capability as well as generation of

126 Applications of RNA-Seq and Omics Strategies - From Microorganisms to Human Health

Genome editing tools have emerged that allow efficient and precise genome manipulation of many organisms including livestock. The genome editing technique is built on engineered, programmable and highly specific nucleases that induce site-specific changes in the genomes of cellular organisms [206]. Subsequent cellular DNA repair processes generates desired insertions, deletions or substitutions at the loci of interest establishing linkages between genetic variations and biological phenotypes [207]. Presently, four artificially engineered nuclease systems have been developed for genome editing: meganucleases derived from microbial mobile elements, zinc finger nucleases (ZFNs) based on eukaryotic transcription factor DNA binding motif, transcription activator-like effector-based nucleases (TALEN) derived from a plan-invasive bacterial protein, and clustered regularly interspaced short palindromic repeats (CRISPR)- CRISPR associated protein 9 (Cas9) system [208]. Centromere and Promoter Factor 1 (Cpf1) is used as an alternative to Cas9 nuclease which requires only a single CRISPR RNA (crRNA) for targeting [209]. CRISPR/Cas9 is easily applicable and has developed really fast over the past

years since only programmable RNA is required to generate sequence specificity [210].

CRISPR–Cas9 system is based on a bacterial CRISPR-Cas9 nuclease from *Streptococcus pyogenes* enabling inexpensive and high-throughput interrogation of gene function [211]. CRISPR-based screening can be used to study non-coding sequences, characterize enhancer elements and regulatory sequences crucial to elucidate the roles of ncRNA [212]. With the CRISPR–Cas9 system, the genome can be sliced at specific sites [213]. Genome editing techniques have been modified and used to alter the genomes of many organisms, thus offering opportunities for generation of genetically modified farm animals [214]. CRISPR offers the ability to target and study particular DNA sequences in the vast expanse of a genome [215]. There are two chief ingredients in the CRISPR–Cas9 system: a Cas9 enzyme that snips through DNA like a pair of molecular scissors, and a small RNA molecule that directs the scissors to a specific sequence of DNA to make the cut. The genome can be edited as desired at nearly any site if a template is provided [216].

In order to adapt this far-reaching application of gene-editing technology to agricultural improvement, various approaches have been applied to a number of livestock species. In pigs, direct cytoplasmic injection of Cas9 mRNA and single-guide RNA into zygotes generated biallelic knockout piglets [217]. The CRISPR-Cas9 system was used to generate gene-edited pigs protected from porcine reproductive and respiratory syndrome virus [218] and to genetically modify single blastocyst inducing indel mutations in a given gene locus[219]. Both Talen and ZNF have been injected directly into pig zygotes to produce live genome edited pigs [220]. Similarly, the porcine myostatin (MSTN) gene, which functions as a negative regulator of muscle growth, was

new biomedical models [205].

With the application of next generation sequencing technologies, the number of ncRNAs reported in livestock species has increased dramatically in the last 5 years. Various tools and pipelines have been introduced to make sense out of ncRNA sequence data. This chapter has provided a comprehensive overview of the current and emerging tools and methods for generating and analyzing ncRNA (miRNA, lncRNA as well as other small ncRNAs) sequence data (transcriptome) with special emphases on the tools that can be applied to livestock species. While bioinformatics tools for miRNA analyses are quite mature, there is a general lack of comprehensive bioinformatics tools for lncRNA and other small ncRNAs. It is our belief that comprehensive "omics" databases that integrate existing and future ncRNA transcriptome databases in the framework of livestock species will contribute towards elucidation of the ambiguity surrounding RNA sequence data. Moreover, given the fact that several emerging platforms (such as genome editing tools) for understanding ncRNAs have been introduced recently, these tools certainly bring great opportunities for broader and also deeper exploration of ncRNA functions. In addition, meticulous *in silico* prediction and careful interpretation of results are critical when handling ncRNA sequence data. Finally, wet-lab validation of the results of transcriptome data will be vital to confirm the functions of ncRNAs in livestock species.

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