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CRISPR-on for activation of endogenous SMARCA4 and TFAP2C expression in bovine embryos.
Reproduction. 2020 06; 159(6):767-778.R

Abstract

CRISPR-mediated transcriptional activation, also known as CRISPR-on, has proven efficient for activation of individual or multiple endogenous gene expression in cultured cells from several species. However, the potential of CRISPR-on technology in preimplantation mammalian embryos remains to be explored. Here, we report for the first time the successful modulation of endogenous gene expression in bovine embryos by using the CRISPR-on system. As a proof of principle, we targeted the promoter region of either SMARCA4 or TFAP2C genes, transcription factors implicated in trophoblast lineage commitment during embryo development. We demonstrate that CRISPR-on provides temporal control of endogenous gene expression in bovine embryos, by simple cytoplasmic injection of CRISPR RNA components into one cell embryos. dCas9VP160 activator was efficiently delivered and accurately translated into protein, being detected in the nucleus of all microinjected blastomeres. Our approach resulted in the activation of SMARCA expression shortly after microinjection, with a consequent effect on downstream differentiation promoting factors, such as TFAP2C and CDX2. Although targeting of TFAP2C gene did not result in a significant increase in TFAP2C expression, there was a profound induction in CDX2 expression on day 2 of development. Finally, we demonstrate that CRISPR-on system is suitable for gene expression modulation during the preimplantation period, since no detrimental effect was observed on microinjected embryo development. This study constitutes a first step toward the application of the CRISPR-on system for the study of early embryo cell fate decisions in cattle and other mammalian embryos, as well as to design novel strategies that may lead to an improved trophectoderm development.

Authors+Show Affiliations

Laboratorio de Biotecnología Animal, Facultad de Agronomía, Universidad de Buenos Aires/INPA CONICET, CABA, Argentina.Laboratorio de Biotecnología Animal, Facultad de Agronomía, Universidad de Buenos Aires/INPA CONICET, CABA, Argentina.Laboratorio de Biotecnología Animal, Facultad de Agronomía, Universidad de Buenos Aires/INPA CONICET, CABA, Argentina.Laboratorio de Biotecnología Animal, Facultad de Agronomía, Universidad de Buenos Aires/INPA CONICET, CABA, Argentina.Instituto de Biotecnología, CICVyA, INTA, Hurlingam, Buenos Aires, Argentina.Centro de Ciencias Veterinarias, Universidad Maimónides, CABA, Argentina.Laboratorio de Biotecnología Animal, Facultad de Agronomía, Universidad de Buenos Aires/INPA CONICET, CABA, Argentina.Department of Animal Science, Michigan State University, East Lansing, Michigan, USA.Laboratorio de Biotecnología Animal, Facultad de Agronomía, Universidad de Buenos Aires/INPA CONICET, CABA, Argentina. Seung Kim Lab, Department of Developmental Biology, Stanford University School of Medicine, Stanford, California, USA.Laboratorio de Biotecnología Animal, Facultad de Agronomía, Universidad de Buenos Aires/INPA CONICET, CABA, Argentina.

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

32240977

Citation

Savy, Virginia, et al. "CRISPR-on for Activation of Endogenous SMARCA4 and TFAP2C Expression in Bovine Embryos." Reproduction (Cambridge, England), vol. 159, no. 6, 2020, pp. 767-778.
Savy V, Alberio V, Canel NG, et al. CRISPR-on for activation of endogenous SMARCA4 and TFAP2C expression in bovine embryos. Reproduction. 2020;159(6):767-778.
Savy, V., Alberio, V., Canel, N. G., Ratner, L. D., Gismondi, M. I., Ferraris, S. F., Fernandez-Martín, R., Knott, J. G., Bevacqua, R. J., & Salamone, D. F. (2020). CRISPR-on for activation of endogenous SMARCA4 and TFAP2C expression in bovine embryos. Reproduction (Cambridge, England), 159(6), 767-778. https://doi.org/10.1530/REP-19-0517
Savy V, et al. CRISPR-on for Activation of Endogenous SMARCA4 and TFAP2C Expression in Bovine Embryos. Reproduction. 2020;159(6):767-778. PubMed PMID: 32240977.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - CRISPR-on for activation of endogenous SMARCA4 and TFAP2C expression in bovine embryos. AU - Savy,Virginia, AU - Alberio,Virgilia, AU - Canel,Natalia G, AU - Ratner,Laura D, AU - Gismondi,Maria I, AU - Ferraris,Sergio F, AU - Fernandez-Martín,Rafael, AU - Knott,Jason G, AU - Bevacqua,Romina J, AU - Salamone,Daniel F, PY - 2019/10/30/received PY - 2020/04/02/accepted PY - 2020/4/3/pubmed PY - 2020/4/3/medline PY - 2020/4/3/entrez SP - 767 EP - 778 JF - Reproduction (Cambridge, England) JO - Reproduction VL - 159 IS - 6 N2 - CRISPR-mediated transcriptional activation, also known as CRISPR-on, has proven efficient for activation of individual or multiple endogenous gene expression in cultured cells from several species. However, the potential of CRISPR-on technology in preimplantation mammalian embryos remains to be explored. Here, we report for the first time the successful modulation of endogenous gene expression in bovine embryos by using the CRISPR-on system. As a proof of principle, we targeted the promoter region of either SMARCA4 or TFAP2C genes, transcription factors implicated in trophoblast lineage commitment during embryo development. We demonstrate that CRISPR-on provides temporal control of endogenous gene expression in bovine embryos, by simple cytoplasmic injection of CRISPR RNA components into one cell embryos. dCas9VP160 activator was efficiently delivered and accurately translated into protein, being detected in the nucleus of all microinjected blastomeres. Our approach resulted in the activation of SMARCA expression shortly after microinjection, with a consequent effect on downstream differentiation promoting factors, such as TFAP2C and CDX2. Although targeting of TFAP2C gene did not result in a significant increase in TFAP2C expression, there was a profound induction in CDX2 expression on day 2 of development. Finally, we demonstrate that CRISPR-on system is suitable for gene expression modulation during the preimplantation period, since no detrimental effect was observed on microinjected embryo development. This study constitutes a first step toward the application of the CRISPR-on system for the study of early embryo cell fate decisions in cattle and other mammalian embryos, as well as to design novel strategies that may lead to an improved trophectoderm development. SN - 1741-7899 UR - https://www.unboundmedicine.com/medline/citation/32240977/CRISPR-on_for_activation_of_endogenous_SMARCA4_and_TFAP2C_expression_in_bovine_embryos L2 - https://rep.bioscientifica.com/doi/10.1530/REP-19-0517 DB - PRIME DP - Unbound Medicine ER -
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