One-Step In Vitro Generation of ETV2-Null Pig Embryos

Abstract

Each year, tens of thousands of people worldwide die of end-stage organ failure due to the limited availability of organs for use in transplantation. To meet this clinical demand, one of the last frontiers of regenerative medicine is the generation of humanized organs in pigs from pluripotent stem cells (PSCs) via blastocyst complementation. For this, organ-disabled pig models are needed. As endothelial cells (ECs) play a critical role in xenotransplantation rejection in every organ, we aimed to produce hematoendothelial-disabled pig embryos targeting the master transcription factor ETV2 via CRISPR-Cas9-mediated genome modification. In this study, we designed five different guide RNAs (gRNAs) against the DNA-binding domain of the porcine ETV2 gene, which were tested on porcine fibroblasts in vitro. Four out of five guides showed cleavage capacity and, subsequently, these four guides were microinjected individually as ribonucleoprotein complexes (RNPs) into one-cell-stage porcine embryos. Next, we combined the two gRNAs that showed the highest targeting efficiency and microinjected them at higher concentrations. Under these conditions, we significantly improved the rate of biallelic mutation. Hence, here, we describe an efficient one-step method for the generation of hematoendothelial-disabled pig embryos via CRISPR-Cas9 microinjection in zygotes. This model could be used in experimentation related to the in vivo generation of humanized organs.

Keywords: CRISPR/Cas9; ETV2; gene editing; porcine embryos; vascular development.

Figure 1

(a) Representation of the pig ETV2 gene structure; The ETS-binding domain is encoded by exon 5 and 6. (b) The genomic target sequence and top five guides designed to target the ETV2 exon 5; (c) Analysis of the Surveyor digestion showing that guide RNAs #1 to #4 are functional in vitro while guide 5 did not mediate the Cas9 activity; specific bands are highlighted with green asterisks. One band at 200 bp that does not correspond to CRISPR/Cas9 gene editing is present in all pig fibroblast samples, including the negative control (−); red asterisks. C+: kit’s GC heterodimer and C−: CC homodimer are included as positive and negative control of Surveyor activity, respectively; (d) an SNP in the genomic DNA of porcine fibroblasts was discovered by comparing the sequences of 9 different PCR samples from control fibroblasts. The SNP (C/G) is shown with colors. Its position on ETV2 explains the 200-bp band produced by Surveyor analysis.

Figure 2

(a) Phase contrast microscope photography of a zygote during the process of CRISPR/Cas9 microinjection (upper panel) and microinjected embryos that reached the blastocyst stage upon in vitro culture (bottom panel). Scale bars: 100 μm; (b) bar graphs showing the percentage of blastocysts edited in the ETV2 gene upon Cas9/gRNA microinjection (with individual or dual gRNAs). Orange bars indicate the percentage of mosaic gene editing and purple bars indicate the percentage of biallelic gene editing; (c) deletion obtained with gRNA1; (d) indels obtained with combined gRNA1 and gRNA4 microinjection at a 10X concentration. Gray rectangles indicate the guide homology regions. The PAM sequence is labeled in orange. The purple arrows mark the cleavage site position and corresponds to base 0.