60 CRISPR-on, a new tool for activation of endogenous gene expression in bovine embryos

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 invitro, making possible the modulation of the cell-differentiation state. However, CRISPR-mediated transcriptional activation (CRISPR-on) has been mainly used invitro, 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 invitro-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