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Article ; Online: Protocol for the electroporation of CRISPR-Cas for DNA and RNA targeting in Bos taurus zygotes.

Biase, Fernando H / Schettini, Gustavo

STAR protocols

2024 Volume 5, Issue 1, Page(s) 102940

Abstract: The use of CRISPR-Cas9 ribonucleoproteins has revolutionized manipulation of genomes. Here, we present a protocol for the electroporation of CRISPR-Cas for DNA and RNA targeting in Bos taurus zygotes. First, we describe steps for production and ... ...

Abstract	The use of CRISPR-Cas9 ribonucleoproteins has revolutionized manipulation of genomes. Here, we present a protocol for the electroporation of CRISPR-Cas for DNA and RNA targeting in Bos taurus zygotes. First, we describe steps for production and preparation of presumptive zygotes for electroporation. The first electroporation introduces ribonucleoproteins formed by Cas9D10A with two guide RNAs to target DNA, and the second introduces the same ribonucleoprotein complex to target DNA plus Cas13a with one guide RNA to target RNAs. For complete details on the use and execution of this protocol, please refer to Nix et al.
MeSH term(s)	Cattle ; Animals ; Zygote ; CRISPR-Cas Systems/genetics ; Gene Editing/methods ; RNA, Guide, CRISPR-Cas Systems ; RNA/genetics ; Electroporation/methods ; DNA/genetics ; Ribonucleoproteins/genetics
Chemical Substances	RNA, Guide, CRISPR-Cas Systems ; RNA (63231-63-0) ; DNA (9007-49-2) ; Ribonucleoproteins
Language	English
Publishing date	2024-03-08
Publishing country	United States
Document type	Journal Article
ISSN	2666-1667
ISSN (online)	2666-1667
DOI	10.1016/j.xpro.2024.102940
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Article ; Online: Isolation of high-quality total RNA and RNA sequencing of single bovine oocytes.

Biase, Fernando H

STAR protocols

2021 Volume 2, Issue 4, Page(s) 100895

Abstract: ... of this protocol, please refer to Biase and Kimble (2018). ...

Abstract	Studying individual mammalian oocytes has been extremely valuable for the understanding of the molecular composition of oocytes including RNA storage. Here, a detailed protocol for isolation of oocytes, extraction of total RNA from single oocytes followed by full-length cDNA amplification, and library preparation is presented. The procedure permits the production of cost-effective and high-quality sequencing libraries. This protocol can be adapted for transcriptome analysis of oocytes from other species and be used to generate high-quality data from single embryos. For complete details on the use and execution of this protocol, please refer to Biase and Kimble (2018).
MeSH term(s)	Animals ; Cattle ; Computational Biology/methods ; DNA, Complementary/genetics ; DNA, Complementary/metabolism ; Female ; Gene Expression Profiling/methods ; Oocytes/metabolism ; RNA/genetics ; RNA/metabolism ; Sequence Analysis, RNA/methods ; Transcriptome/genetics
Chemical Substances	DNA, Complementary ; RNA (63231-63-0)
Language	English
Publishing date	2021-10-19
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ISSN	2666-1667
ISSN (online)	2666-1667
DOI	10.1016/j.xpro.2021.100895
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Article ; Online: A multi-omics analysis identifies molecular features associated with fertility in heifers (Bos taurus).

Marrella, Mackenzie A / Biase, Fernando H

Scientific reports

2023 Volume 13, Issue 1, Page(s) 12664

Abstract: Infertility or subfertility is a critical barrier to sustainable cattle production, including in heifers. The development of heifers that do not produce a calf within an optimum window of time is a critical factor for the profitability and sustainability ...

Abstract	Infertility or subfertility is a critical barrier to sustainable cattle production, including in heifers. The development of heifers that do not produce a calf within an optimum window of time is a critical factor for the profitability and sustainability of the cattle industry. In parallel, heifers are an excellent biomedical model for understanding the underlying etiology of infertility because well-nourished heifers can still be infertile, mostly because of inherent physiological and genetic causes. Using a high-density single nucleotide polymorphism (SNP) chip, we collected genotypic data, which were analyzed using an association analysis in PLINK with Fisher's exact test. We also produced quantitative transcriptome data and proteome data. Transcriptome data were analyzed using the quasi-likelihood test followed by the Wald's test, and the likelihood test and proteome data were analyzed using a generalized mixed model and Student's t-test. We identified two SNPs significantly associated with heifer fertility (rs110918927, chr12: 85648422, P = 6.7 × 10
MeSH term(s)	Cattle ; Female ; Animals ; Multiomics ; Proteome ; Fertility/genetics ; Infertility ; Genotype
Chemical Substances	Proteome
Language	English
Publishing date	2023-08-04
Publishing country	England
Document type	Journal Article ; Research Support, Non-U.S. Gov't ; Research Support, U.S. Gov't, Non-P.H.S.
ZDB-ID	2615211-3
ISSN	2045-2322 ; 2045-2322
ISSN (online)	2045-2322
ISSN	2045-2322
DOI	10.1038/s41598-023-39858-0
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Article: Robust identification of regulatory variants (eQTLs) using a differential expression framework developed for RNA-sequencing.

Marrella, Mackenzie A / Biase, Fernando H

Journal of animal science and biotechnology

2023 Volume 14, Issue 1, Page(s) 62

Abstract: Background: A gap currently exists between genetic variants and the underlying cell and tissue biology of a trait, and expression quantitative trait loci (eQTL) studies provide important information to help close that gap. However, two concerns that ... ...

Abstract	Background: A gap currently exists between genetic variants and the underlying cell and tissue biology of a trait, and expression quantitative trait loci (eQTL) studies provide important information to help close that gap. However, two concerns that arise with eQTL analyses using RNA-sequencing data are normalization of data across samples and the data not following a normal distribution. Multiple pipelines have been suggested to address this. For instance, the most recent analysis of the human and farm Genotype-Tissue Expression (GTEx) project proposes using trimmed means of M-values (TMM) to normalize the data followed by an inverse normal transformation. Results: In this study, we reasoned that eQTL analysis could be carried out using the same framework used for differential gene expression (DGE), which uses a negative binomial model, a statistical test feasible for count data. Using the GTEx framework, we identified 35 significant eQTLs (P < 5 × 10 Conclusions: Our results show that transforming RNA-sequencing data to fit a normal distribution prior to eQTL analysis is not required when the DGE framework is employed. Our proposed approach detected biologically relevant variants that otherwise would not have been identified due to data transformation to fit a normal distribution.
Language	English
Publishing date	2023-05-05
Publishing country	England
Document type	Journal Article
ZDB-ID	2630162-3
ISSN	2049-1891 ; 1674-9782
ISSN (online)	2049-1891
ISSN	1674-9782
DOI	10.1186/s40104-023-00861-0
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Article ; Online: Isolation of high-quality total RNA and RNA sequencing of single bovine oocytes

Fernando H. Biase

STAR Protocols, Vol 2, Iss 4, Pp 100895- (2021)

2021

Abstract: ... of this protocol, please refer to Biase and Kimble (2018). ...

Abstract	Summary: Studying individual mammalian oocytes has been extremely valuable for the understanding of the molecular composition of oocytes including RNA storage. Here, a detailed protocol for isolation of oocytes, extraction of total RNA from single oocytes followed by full-length cDNA amplification, and library preparation is presented. The procedure permits the production of cost-effective and high-quality sequencing libraries. This protocol can be adapted for transcriptome analysis of oocytes from other species and be used to generate high-quality data from single embryos.For complete details on the use and execution of this protocol, please refer to Biase and Kimble (2018).
Keywords	Bioinformatics ; Cell isolation ; Gene Expression ; Molecular Biology ; RNAseq ; Sequencing ; Science (General) ; Q1-390
Language	English
Publishing date	2021-12-01T00:00:00Z
Publisher	Elsevier
Document type	Article ; Online
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Article ; Online: Robust identification of regulatory variants (eQTLs) using a differential expression framework developed for RNA-sequencing

Mackenzie A. Marrella / Fernando H. Biase

Journal of Animal Science and Biotechnology, Vol 14, Iss 1, Pp 1-

2023 Volume 11

Abstract: Abstract Background A gap currently exists between genetic variants and the underlying cell and tissue biology of a trait, and expression quantitative trait loci (eQTL) studies provide important information to help close that gap. However, two concerns ... ...

Abstract	Abstract Background A gap currently exists between genetic variants and the underlying cell and tissue biology of a trait, and expression quantitative trait loci (eQTL) studies provide important information to help close that gap. However, two concerns that arise with eQTL analyses using RNA-sequencing data are normalization of data across samples and the data not following a normal distribution. Multiple pipelines have been suggested to address this. For instance, the most recent analysis of the human and farm Genotype-Tissue Expression (GTEx) project proposes using trimmed means of M-values (TMM) to normalize the data followed by an inverse normal transformation. Results In this study, we reasoned that eQTL analysis could be carried out using the same framework used for differential gene expression (DGE), which uses a negative binomial model, a statistical test feasible for count data. Using the GTEx framework, we identified 35 significant eQTLs (P < 5 × 10–8) following the ANOVA model and 39 significant eQTLs (P < 5 × 10–8) following the additive model. Using a differential gene expression framework, we identified 930 and six significant eQTLs (P < 5 × 10–8) following an analytical framework equivalent to the ANOVA and additive model, respectively. When we compared the two approaches, there was no overlap of significant eQTLs between the two frameworks. Because we defined specific contrasts, we identified trans eQTLs that more closely resembled what we expect from genetic variants showing complete dominance between alleles. Yet, these were not identified by the GTEx framework. Conclusions Our results show that transforming RNA-sequencing data to fit a normal distribution prior to eQTL analysis is not required when the DGE framework is employed. Our proposed approach detected biologically relevant variants that otherwise would not have been identified due to data transformation to fit a normal distribution.
Keywords	Differential gene expression ; eQTL analysis ; Gene expression ; RNA-sequencing ; Single nucleotide polymorphism ; Animal culture ; SF1-1100 ; Veterinary medicine ; SF600-1100
Subject code	310
Language	English
Publishing date	2023-05-01T00:00:00Z
Publisher	BMC
Document type	Article ; Online
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Article ; Online: Oocyte Developmental Competence: Insights from Cross-Species Differential Gene Expression and Human Oocyte-Specific Functional Gene Networks.

Biase, Fernando H

Omics : a journal of integrative biology

2017 Volume 21, Issue 3, Page(s) 156–168

Abstract: Understanding oocyte developmental competence remains a key challenge for reproductive biology and systems sciences. The transcriptome of oocytes in eutherians is highly complex and is associated with the success of embryo development. Due to sample ... ...

Abstract	Understanding oocyte developmental competence remains a key challenge for reproductive biology and systems sciences. The transcriptome of oocytes in eutherians is highly complex and is associated with the success of embryo development. Due to sample limitations from humans, animal models are used for investigation of the oocyte transcriptome. Nonetheless, little is known about the diversity of the oocyte transcriptome across eutherians. In this report, comprehensive investigation of 7 public data sets in 4 species, human, macaque, mice, and cattle, shows that 16,572 genes are expressed in oocytes. Approximately 26% of the genes were expressed in all four species. There were 1390, 489, and 187 genes specifically expressed in human, mice, and cattle oocytes, respectively. Coexpression clustering of the genes specifically expressed in human oocytes revealed functional enrichment (FDR <0.05) of Gene Ontology (GO) terms important for oocyte physiology (i.e., "cellular response to metal ion," "negative regulation of growth," "hormone activity," and "receptor activity"). Interrogation of 4 data sets revealed 26 genes whose expressions were significantly (FDR ≤0.1) associated with oocyte developmental competence and concordant fold change in 2 studies. The genes AK2, AKAP1, ECHS1, MRPL10, MRPL24, PTRH2, STX17, SUCLG1, SUOX, and TOMM34 were associated with the GO term "mitochondrion" (FDR <0.01). Collectively, the results offer new insights on gene transcript levels associated with oocyte developmental competence and the central role of mitochondrion for oocyte's health among eutherians. Caution should be exercised, however, when extending the inferences related to gene expression associated with oocyte quality across eutherians.
MeSH term(s)	Female ; Gene Ontology ; Gene Regulatory Networks/genetics ; Humans ; Mitochondria/metabolism ; Oocytes/metabolism ; Oocytes/physiology
Language	English
Publishing date	2017-03
Publishing country	United States
Document type	Journal Article
ZDB-ID	2030312-9
ISSN	1557-8100 ; 1536-2310
ISSN (online)	1557-8100
ISSN	1536-2310
DOI	10.1089/omi.2016.0177
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Article ; Online: The blueprint of RNA storages relative to oocyte developmental competence in cattle (Bos taurus).

Walker, Bailey N / Biase, Fernando H

Biology of reproduction

2020 Volume 102, Issue 4, Page(s) 784–794

Abstract: From the time oocytes leave quiescence, there are constant microenvironmental influences contributing to development, thus acquiring developmental competence is not a simple, linear phenomenon. During folliculogenesis, oocytes experience many ... ...

Abstract	From the time oocytes leave quiescence, there are constant microenvironmental influences contributing to development, thus acquiring developmental competence is not a simple, linear phenomenon. During folliculogenesis, oocytes experience many morphological and cytological changes that contribute toward the acquisition of developmental competence, a process defined by an oocyte's ability to progress through folliculogenesis, be fertilized, undergo cleavage, and develop into an embryo. Many factors, such as ovarian follicle size, cow age, and the morphology of the cumulus-oocyte complex, have been extensively investigated to understand this process. In parallel to aiding in the understanding of oocyte biology, these features have been used to characterize an oocyte's ability to achieve competence. In addition, oocytes undergo intense gene transcription and protein translation to accumulate the maternal stores. When the oocyte is fully grown, most genes are transcriptionally inactive, and the chromatin is densely compacted. More recently, RNA profiling has been used to further define the transcriptional parameters that are associated with oocyte development. Here, focusing on cattle, we provide an overview of the experimental models commonly used to understand the underlying biology related to oocyte developmental competence. We compiled public data and showed that cattle oocytes can express over 15 000 protein-coding genes, suggesting a complex transcriptome landscape. Surprisingly, less than 2% of the expressed genes have been linked to developmental competence. The identification of the gene products that contribute to oocyte development, and understanding their biological function, are a vital component of our quest toward defining oocyte developmental competence at the molecular level.
MeSH term(s)	Animals ; Cattle ; Cumulus Cells/metabolism ; Female ; Oocytes/metabolism ; Oogenesis/physiology ; Ovarian Follicle/metabolism ; RNA/metabolism
Chemical Substances	RNA (63231-63-0)
Language	English
Publishing date	2020-01-16
Publishing country	United States
Document type	Journal Article ; Review
ZDB-ID	1118-6
ISSN	1529-7268 ; 0006-3363
ISSN (online)	1529-7268
ISSN	0006-3363
DOI	10.1093/biolre/ioaa015
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Article: Beef heifer fertility: importance of management practices and technological advancements.

Moorey, Sarah E / Biase, Fernando H

Journal of animal science and biotechnology

2020 Volume 11, Page(s) 97

Abstract: The development of replacement heifers is at the core of cow-calf beef production systems. In 2020, the USDA, National Agricultural Statistics Service reported 5.771 million beef heifers, 500 pounds and over, are under development for cow replacement. A ... ...

Abstract	The development of replacement heifers is at the core of cow-calf beef production systems. In 2020, the USDA, National Agricultural Statistics Service reported 5.771 million beef heifers, 500 pounds and over, are under development for cow replacement. A compilation of data from several studies indicate that between 85% and 95% of these heifers will become pregnant in their first breeding season. Several thousands of heifers being raised for replacement may not deliver a calf on their first breeding season and result in economic losses to cow-calf producers. Many management procedures have been developed to maximize the reproductive potential of beef heifers. Such approaches include, but are not limited to the following: nutritional management for controlled weight gain, identification of reproductive maturity by physiological and morphological indicators, and the implementation of an estrous synchronization program. The implementation of management strategies has important positive impact(s) on the reproductive efficiency of heifers. There are limitations, however, because some heifers deemed ready to enter their first breeding season do not become pregnant. In parallel, genetic selection for fertility-related traits in beef heifers have not promoted major genetic gains on this particular area, most likely due to low heritability of female fertility traits in cattle. Technologies such as antral follicle counting, DNA genotyping and RNA profiling are being investigated as a means to aid in the identification of heifers of low fertility potential. To date, many polymorphisms have been associated with heifer fertility, but no DNA markers have been identified across herds. Antral follicle count is an indication of the ovarian reserve and is an indicator of the reproductive health of a heifer. We have been working on the identification of transcriptome profiles in heifers associated with pregnancy outcome. Our current investigations integrating protein-coding transcript abundance and artificial intelligence have identified the potential for bloodborne transcript abundance to be used as indicators of fertility potential in beef heifers. In summary, there is an ongoing pressure for reducing costs and increasing efficiency in cow-calf production systems, and new technologies can help reduce the long-standing limitations in beef heifer fertility.
Language	English
Publishing date	2020-10-01
Publishing country	England
Document type	Journal Article ; Review
ZDB-ID	2630162-3
ISSN	2049-1891 ; 1674-9782
ISSN (online)	2049-1891
ISSN	1674-9782
DOI	10.1186/s40104-020-00503-9
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Article ; Online: Ablation of

Nix, Jada L / Schettini, Gustavo P / Speckhart, Savannah L / Ealy, Alan D / Biase, Fernando H

PNAS nexus

2023 Volume 2, Issue 11, Page(s) pgad343

Abstract: CRISPR-Cas ribonucleoproteins (RNPs) are important tools for gene editing in preimplantation embryos. However, the inefficient production of biallelic deletions in cattle zygotes has hindered mechanistic studies of gene function. In addition, the ... ...

Abstract	CRISPR-Cas ribonucleoproteins (RNPs) are important tools for gene editing in preimplantation embryos. However, the inefficient production of biallelic deletions in cattle zygotes has hindered mechanistic studies of gene function. In addition, the presence of maternal RNAs that support embryo development until embryonic genome activation may cause confounding phenotypes. Here, we aimed to improve the efficiency of biallelic deletions and deplete specific maternal RNAs in cattle zygotes using CRISPR-Cas editing technology. Two electroporation sessions with Cas9D10A RNPs targeting exon 1 and the promoter of
Language	English
Publishing date	2023-10-20
Publishing country	England
Document type	Journal Article
ISSN	2752-6542
ISSN (online)	2752-6542
DOI	10.1093/pnasnexus/pgad343
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