Progress on genome-wide CRISPR/Cas9 screening for functional genes and regulatory elements.

The CRISPR/Cas9 system is a powerful tool which has been extensively used for genome editing in the past few years. Nuclease-dead Cas9 (CRISPR/dCas9), a Cas9 protein mutant without splicing ability, along with loss-of- function (LOF), gain-of-function (GOF), or non-coding genes scanning approaches can reveal genome-scale functional determinants. CRISPR/Cas9 has been widely adopted to decipher disease mechanisms and pinpoint drug targets in the life science field, and also provide novel insights into animal genetics and breeding. In this review, we summarize the research progress in high-throughput CRISPR/Cas9 screening for revealing the functional genes and regulatory elements in the whole genome. We also highlight the applications of CRISPR/Cas9 system in the animal cells, providing a reference for gene editing and other related research in related fields.

Systematic identification and characterization of porcine snoRNAs: structural, functional and developmental insights.

Small nucleolar RNAs (snoRNAs) are 50- to 300-nt non-coding RNAs that are involved in critical cellular events, including rRNA/snRNA modification and splicing, ribosome genesis, telomerase formulation and cell proliferation. The identification of snoRNAs in the pig, which is a widely consumed commercial organism that also has important functions in medicine and biology, will enrich the snoRNA kingdom and provide evolutionary clues about snoRNAs. In this study, we performed a systematic identification of snoRNAs in Sus scrofa and obtained 120 candidate snoRNAs, 65 of which were predicted via sequencing from our constructed cDNA library. The others were obtained by computational screening. The primary structural features examined included the sequence length, GC content, conservation of common box motifs and nucleotide diversity. The results indicate that the primary features of H/ACA box snoRNAs are opposite to those of C/D box snoRNAs. Subsequently, based on chromosomal location and host gene determination, we assigned 91 snoRNAs to nine genome organization modes. Gene duplications and translocations are considered to contribute to the high abundant organization in evolution. Functional information about our novel snoRNAs, such as putative targets, modification sites and guide sequences, was predicted by orthologue alignment. A comparative analysis of predicted targets and possible modified loci on U6 snRNA and 5.8S and 18S rRNAs among five species revealed that targets of snoRNA are conserved among species. Furthermore, we performed a quantitative analysis of six representative snoRNA genes in two pig breeds during different developmental stages. Interestingly, all six snoRNAs from one breed expressed in a similar pattern over the tested time points; however, these same six genes had different expression patterns in the other pig breed. Specifically, expression of all six snoRNAs declined significantly from 65 to 90 days post-coitus (dpc) and then increased slightly during adulthood in Tongcheng pigs, whereas the expression of the same six genes increased slowly from 65 dpc until adulthood in Landrace pigs. This expression pattern suggests that most housekeeping, non-coding RNAs from a single pig breed may be similarly expressed during development. Our study adds to the knowledge about the snoRNA family by providing the first genome-wide study of porcine snoRNAs. The comparative analysis of snoRNAs from different pig breeds gave us evolutionary insight into the function of snoRNAs.

[Genome-wide selective sweep analysis on Large White and Tongcheng pigs].

The production performance of pigs has been significantly improved due to long-term artificial selection, and the specific variation characterizations (selection signatures) emerged from the selected genome regions. Different types of breeds are subjected to different selection intensities and had different selection signatures. Selective sweep analysis is one of major methods to detect the selection signatures. In this study, based on the 60K BeadChip genotyping data of both commercial Large White (n=45) and local Tongcheng pigs (n=45), genetic differentiation coefficient Fst was applied to detect the selection signatures. Using gPLINK software to set quality control standards, a total of 34 304 SNPs were selected for statistical analysis. Fst values between two breeds were estimated with Genepop package and the average Fst value was 0.3209. Setting Fst>0.7036 (1% of total number of Fst values) as selection threshold, 344 SNPs were obtained and SNP location annotation indicated that there were 79 candidate genes (Sus scrofa Build 9). Furthermore, network analysis was performed using Ingenuity Pathway Analysis and the preliminary results suggested that most genes were involved in growth, reproduction, and immune response, such as NCOA6, ERBB4, RUNX2, and APOB genes. The findings from this study will contribute to further identification of candidate genes and causal mutations implying for meat production and disease resistance in pig.

MicroRNA-148a promotes myogenic differentiation by targeting the ROCK1 gene.

MicroRNAs are evolutionarily conserved small RNAs that post-transcriptionally regulate gene expression and have emerged as critical regulators of skeletal muscle development. Here, we identified miR-148a as a novel myogenic microRNA that mediated myogenic differentiation. The expression levels of miR-148a increased during C2C12 myoblast differentiation. Overexpression of miR-148a significantly promoted myogenic differentiation of both C2C12 myoblast and primary muscle cells. Blocking the function of miR-148a with a 2'-O-methylated antisense oligonucleotide inhibitor repressed C2C12 myoblast differentiation. Using a bioinformatics approach, we identified Rho-associated coiled-coil containing protein kinase 1 (ROCK1), a known inhibitor of myogenesis, as a target of miR-148a. A dual-luciferase reporter assay was used to demonstrate that miR-148a directly targeted the 3'-UTR of ROCK1. In addition, the overexpression of miR-148a decreased the protein expression of ROCK1 in C2C12 myoblast and primary muscle cells. Furthermore, ROCK1 inhibition with specific siRNA leaded to accelerated myogenic differentiation progression, underscoring a negative regulatory function of ROCK1 in myogenesis. Therefore, our results revealed a novel mechanism in which miR-148a positively regulates myogenic differentiation via ROCK1 down-regulation.

Molecular characteristics of the porcine TIMD4 gene and its association analysis.

As a member of the T cell immunoglobulin domain and mucin domain (TIM) gene family, TIMD4 plays an important role in the immune response. To understand its function more precisely, we isolated it and analyzed its subcellular localization, expression pattern, and associations. The porcine TIMD4 gene included nine exons and eight introns with an open reading frame of 1086 bp encoding 361 amino acids. It had relatively high levels in liver, lymph, and spleen. The fusion protein was localized mainly in the cytoplasm of pig kidney cells (PK15). The promoter region contained a TATA box and GATA3 consensus sites. A single nucleotide polymorphism was identified in intron 3 of the porcine TIMD4 gene, and analysis indicated that it had significant associations with the 17-day red blood cell count (p = 0.0106), hemoglobin (p = 0.0149), and hematocrit (p = 0.0063) and with 32-day hemoglobin (p = 0.0140).

Molecular characterization of Tob1 in muscle development in pigs.

Cell proliferation is an important biological process during myogenesis. Tob1 encoded a member of the Tob/BTG family of anti-proliferative proteins. Our previous LongSAGE (Long Serial Analysis of Gene Expression) analysis suggested that Tob1 was differentially expressed during prenatal skeletal muscle development. In this study, we isolated and characterized the swine Tob1 gene. Subsequently, we examined Tob1 chromosome assignment, subcellular localization and dynamic expression profile in prenatal skeletal muscle (33, 65 and 90 days post-conception, dpc) from Landrace (lean-type) and Tongcheng pigs (obese-type). The Tob1 gene was mapped to pig chromosome 12 (SSC12). The Tob1 protein was distributed throughout the nucleus and cytoplasm of PK15 cells. During prenatal skeletal muscle development, Tob1 was up-regulated and highly expressed in skeletal muscle at 90 dpc in Tongcheng pigs but peaked at 65 dpc in Landrace pigs. This result suggested that there were different proliferation patterns during myogenesis between Tongcheng and Landrace pigs. During postnatal skeletal muscle development, the expression of Tob1 increased with aging, indicating that the proliferation potential of myoblasts decreased in postnatal muscle development. In tissues of adult wuzhishan miniature pigs, the Tob1 gene was highly expressed in skeletal muscle. The expression of Tob1 was significantly increased at day 6 during C2C12 differentiation time, suggesting a possible role in skeletal muscle development. Therefore, this study indicated that Tob1 perhaps played an important role in skeletal muscle development.

Molecular characterization of the BTG2 and BTG3 genes in fetal muscle development of pigs.

BTG2 and BTG3 are two members of the B-cell translocation gene family with anti-proliferative properties. BTG1 gene in this gene family has been reported to play a key role in muscle growth. In this study, we identified and characterized the porcine BTG2 and BTG3 genes, mapped the two genes to porcine chromosomes, and analyzed their expression differences in the longissimus dorci muscle of 33 dpc (day postconception), 65 dpc and 90 dpc in the lean Landrace and fatty Chinese Tongcheng pig breeds. Expression changes in differentiated C2C12 cells were also investigated with myogenin as internal control. The results showed that the porcine BTG2 and BTG3 genes were mapped on SSC9q21-25 and SSC13q47, respectively. BTG2 gene expressed at high levels in skeletal muscle and heart in both Tongcheng and Landrace pigs whereas BTG3 gene expressed at lower levels in skeletal muscle and heart than in other tissues. Furthermore, BTG3 expressed at higher levels in skeletal muscle of Tonghceng compared with Landrace pig. The expression of BTG2 and BTG3 was significantly different in skeletal muscle among different developmental stages and between the two breeds. Expression analysis in murine myoblast cells showed that both genes were induced in differentiated C2C12 cells, suggesting a role of them in myogenic differentiation. Our study indicated that BTG2 and BTG3, especially BTG3 gene, may be important genes for skeletal muscle growth.