60 CRISPR-on, a new tool for activation of endogenous gene expression in bovine embryos
V. Savy A B , V. Alberio A , N. Canel A , L. Ratner A , M. Gismondi C , O. Taboga C , S. Ferraris D , S. Rulli E , R. Fernandez-Martin A , R. Bevacqua A F and D. Salamone AA Laboratorio de Biotecnología Animal, FAUBA/INPA-CONICET, CABA, Buenos Aires, Argentina;
B Reproductive Medicine Group RDBL, NIEHS-NIH, Durham, NC, USA;
C Instituto de Biotecnología, CICVyA, INTA, Hurlingam, Buenos Aires, Argentina;
D CIDME, Universidad Maimónides, CABA, Buenos Aires, Argentina;
E IByME CONICET, CABA, Buenos Aires, Argentina;
F Seung Kim Lab, Department of Developmental Biology, Stanford University School of Medicine, Stanford, CA, USA
Reproduction, Fertility and Development 32(2) 155-155 https://doi.org/10.1071/RDv32n2Ab60
Published: 2 December 2019
Abstract
The CRISPR-Cas9 system enables precise genome editing in mammalian somatic cells and embryos at a very high efficiency. A modified version of Cas9 (dCas9) was engineered, resulting in a DNA binding protein capable of site-specific target recognition but unable to cut the DNA. By means of dCas9 fusion to heterologous domains, including transcriptional activators or repressors, specific modulation of gene expression has successfully been achieved in vitro, making possible the modulation of the cell-differentiation state. However, CRISPR-mediated transcriptional activation (CRISPR-on) has been mainly used in vitro, and to our knowledge, there are no reports regarding its use for the activation of endogenous gene expression in mammalian embryos. As a proof of principle, we evaluated the CRISPR-on system in bovine embryos for modulation of endogenous expression of SMARCA4 and TFAP2C, transcription factors implicated in trophoblast lineage commitment. We hypothesised that CRISPR-on may induce SMARCA4 or TFAP2C endogenous expression, enabling the design of strategies to induce trophectoderm proliferation of in vitro-derived embryos. To this aim, we designed and synthesised 4 non-overlapping single guide RNAs to target the regulatory region of each of these target genes. Presumptive zygotes were cytoplasmically microinjected with a mix containing dCas9-VP160 mRNA and a pool of 4 single guide RNAs targeting SMARCA4 (dCas9_SM group) or TFAP2C (dCas9_TF group). As control, a non-injected group was also included. Analysis was carried out in pools of 10 early embryos or 5 blastocysts and at least 3 biological replicates were included. Gene expression was assessed by RTqPCR at Days 2, 4, and 7 after microinjection and data were normalized to that obtained for the non-injected group. The CRISPR-on system was efficient to induce expression of SMARCA4 two days after microinjection (dCas9_SM group, Mann-Whitney t-test; P < 0.05), but failed to significantly increase TFAP2C expression (dCas9_TF group). Surprisingly, CDX2, which is a downstream effector for trophectoderm maintenance, was induced both in dCas9_SM and dCas9_TF groups, supporting the CRISPR-mediated induction of targeted transcription factors. However, no changes were observed in the endogenous level of NANOG. Additional analysis is currently ongoing to determine whether CRISPR-on mediated induction of SMARCA4 and/or TFAP2C expression affects lineage specification and regulation. To our knowledge, this is the first report on the use of CRISPR-on for modulation of endogenous gene expression in mammalian embryos. Our study lays the foundations for CRISPR-on application in embryos as a useful tool to understand key cell fate decisions and will enable unprecedented studies of significance to embryo development, cell differentiation, and segregation.
