201 Testing of single guide RNAs, optimization of transfection, and selection systems for the generation of SRY knockout foetal fibroblast cells

Abstract

Obtaining male and female cells from a male could have important implications for endangered mammalian species and domestic animal production. Achieving this could constitute a proof of concept of the use of assisted reproduction technologies for the conservation of endangered species. In particular, SRY is the principal male sex determinant gene and is found on the short arm of the Y chromosome. With the introduction of nuclease-mediated genome editing technologies, such as the CRISPR/Cas9 system, it is possible to envision precise DNA targeting in this gene as strategy to manipulate the sex of cell lines. Based on the above, we tested the CRISPR/Cas9 system to later create knockout (KO) cell lines of the SRY gene in bovine. The aim of this work was (1) to test the efficiency of single guide (sg)RNAs designed to target the bovine SRY gene in HEK293T cell line, and (2) to optimize blasticidin concentrations and electroporation conditions in bovine fetal fibroblast (BFF). To test sgRNA efficiency, we used 3 sgRNA designed and tested over the sequence of bovine SRY gene (690 bp). The efficiency of each sgRNA was evaluated in a heterologous way by using a modification of pCAG-EGxxFP (plasmid #50716, Addgene, Cambridge, MA, USA) in which we inserted the bovine SRY sequence (NCBI Reference Sequence: NC_016145.1), and that only results in green fluorescent protein (GFP) expression upon cutting by the CRISPR/Cas9 system. We transfected the HEK293T cell line with the following groups to assess the efficiency of 3 sgRNA: (1) btSRY1 + Cas9 + pCAG-EGSRYFP; (2) btSRY2 + Cas9 + pCAG-EGSRYFP; (3) btSRY3 + Cas9 + pCAG-EGSRYFP; (4) PU6-empty + Cas9 + pCAG- EGSRYFP; (5) GFP-only control. In addition, 6 blasticidin concentrations (0, 1, 2.5, 5, 10, and 20 µg mL⁻¹) and 3 electroporation conditions (Tfx1, Tfx2, and Tfx3) were tested, both in BFF. Electroporation conditions were as follows: Tfx1 = 1.25 kV cm⁻¹; number of pulses = 3; electrode gap = 4 mm; interval = 100 ms; Tfx2 = 1.2 kV/cm; number of pulses = 4; electrode gap = 4 mm; interval = 100 ms; and Tfx3 = voltage: 350V, LV mode; pulse length = 100 µs; number of pulses = 4; electrode gap = 4 mm; interval = 100 ms. Statistical analyses were performed using 2-tailed Mann-Whitney tests. Results for sgRNA efficiency, based on GFP expression by counting GFP+ cells under fluorescent microscopy showed that btSRY1 (17%) and btSRY2 (13%) worked significantly better than btSRY3 (1%). The blasticidin selection assay showed that 5, 10, and 20 µg mL⁻¹were significantly more lethal than 0, 1 and 2.5 µg mL⁻¹, by counting living cells in Neubauer chamber. We chose 5 µg mL⁻¹ as the concentration for future experiments. Last, the Tfx2 electroporation protocol (11.34%) was more efficient than the other 2 protocols tested (Tfx1 and Tfx3: 3.48 and 0.86%, respectively), based on the counting of GFP+ cells under fluorescent microscopy. Electroporation of BFF with btSRY1, btSRY2, or both and Cas9 using Tfx2 protocol and molecular characterisation of colonies are currently in progress with the ultimate objective of producing SRY knockout bovine embryos by somatic cell nuclear transfer.