**6. Emerging platforms and technologies for understanding and using ncRNAs**

Efficient and reliable techniques for accurate detection of genome information are important for productivity and health of livestock species [202]. The introduction of next generation 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 new biomedical models [205].

disrupted using CRISPR/Cas9 system to efficiently generate biologically safe genetically modified pigs [221]. Similarly, zygote injection of TALEN mRNA targeting MSTN gene led to produc-

Transcriptome Analysis of Non‐Coding RNAs in Livestock Species: Elucidating the Ambiguity

http://dx.doi.org/10.5772/intechopen.69872

127

In cattle, the CRISPR/Cas9 system was successfully used to clone embryos that could be used to develop livestock transgenes for agricultural science [222]. Hornlessness was introduced into dairy cattle by genome editing and reproductive cloning providing the potential to improve the welfare of millions of cattle [223]. In the cattle industry, gene-edited calves have been produced with specified genetics by ovum pickup, *in vitro* fertilization and zygote microinjection (OPU-IVF-ZM). The CRISPR/Cas9 system has also been used efficiently to gen-

In livestock, CRISPR-Cas9 has been greatly enhanced by single-guide RNA generating sitespecific DNA breaks through homology-directed repair and used for diverse applications, from disease modelling of individual loci to parallelized loss-of-function screens of thousands of regulatory elements [225]. Equally, bioinformatics designs for CRISPR deletions are now possible with a tool known as CRISPETa developed with efficient CRISPR deletion of an enhancer and exonic fragment of MALAT1, a lncRNA. CRISPETa can be used for single target regions or thousands of targets and has high-coverage library designs for entire classes of non-coding elements which can be adopted for use in livestock species [226]. CRISPR-Cas9 may be used with a gene drive incorporated with genome edit to investigate the control of any biological process and can be used to accelerate livestock breeding [225]. Gene drives can be constructed with the use of CRISPR-Cas9 tool that can favour the inheritance of edited alleles possible to modify a whole population [227]. In the DNA, a double strand break can be initiated by a gene drive during the copying process. Using the sequence of the chromosome containing the gene drive elements as a repair template, the DNA break could be repaired by cellular pathways such as homology-directed repair [228]. Editing the genomic DNA elements targeting non-coding regions is vital since silencing of ncRNA genes using RNA interference tools still presents major challenges. An improved vector system adapted to delete non-protein-coding regulatory elements; double excision CRISPR Knockout (DECKO) using two-step cloning to produce vectors (lentivirus) with two guide RNAs concurrently [229], has been used effectively to silenced five ncRNAs (miRNAs-miR21, miR29a and lncRNAs-UCA1 and MALAT1) [230]. The use of genome editing technologies will create novel viewpoints for enquiry to advance our knowledge on biological function of ncRNAs in livestock species and

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

tion of gene-edited cattle and sheep [205]

erate gene knock out sheep [224].

facilitate creating animals with precise alterations.

**7. Conclusion and remarks**

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 disrupted using CRISPR/Cas9 system to efficiently generate biologically safe genetically modified pigs [221]. Similarly, zygote injection of TALEN mRNA targeting MSTN gene led to production of gene-edited cattle and sheep [205]

In cattle, the CRISPR/Cas9 system was successfully used to clone embryos that could be used to develop livestock transgenes for agricultural science [222]. Hornlessness was introduced into dairy cattle by genome editing and reproductive cloning providing the potential to improve the welfare of millions of cattle [223]. In the cattle industry, gene-edited calves have been produced with specified genetics by ovum pickup, *in vitro* fertilization and zygote microinjection (OPU-IVF-ZM). The CRISPR/Cas9 system has also been used efficiently to generate gene knock out sheep [224].

In livestock, CRISPR-Cas9 has been greatly enhanced by single-guide RNA generating sitespecific DNA breaks through homology-directed repair and used for diverse applications, from disease modelling of individual loci to parallelized loss-of-function screens of thousands of regulatory elements [225]. Equally, bioinformatics designs for CRISPR deletions are now possible with a tool known as CRISPETa developed with efficient CRISPR deletion of an enhancer and exonic fragment of MALAT1, a lncRNA. CRISPETa can be used for single target regions or thousands of targets and has high-coverage library designs for entire classes of non-coding elements which can be adopted for use in livestock species [226]. CRISPR-Cas9 may be used with a gene drive incorporated with genome edit to investigate the control of any biological process and can be used to accelerate livestock breeding [225]. Gene drives can be constructed with the use of CRISPR-Cas9 tool that can favour the inheritance of edited alleles possible to modify a whole population [227]. In the DNA, a double strand break can be initiated by a gene drive during the copying process. Using the sequence of the chromosome containing the gene drive elements as a repair template, the DNA break could be repaired by cellular pathways such as homology-directed repair [228]. Editing the genomic DNA elements targeting non-coding regions is vital since silencing of ncRNA genes using RNA interference tools still presents major challenges. An improved vector system adapted to delete non-protein-coding regulatory elements; double excision CRISPR Knockout (DECKO) using two-step cloning to produce vectors (lentivirus) with two guide RNAs concurrently [229], has been used effectively to silenced five ncRNAs (miRNAs-miR21, miR29a and lncRNAs-UCA1 and MALAT1) [230]. The use of genome editing technologies will create novel viewpoints for enquiry to advance our knowledge on biological function of ncRNAs in livestock species and facilitate creating animals with precise alterations.
