Generation of myostatin edited horse embryos using CRISPR/Cas9 technology and somatic cell nuclear transfer.

The application of new technologies for gene editing in horses may allow the generation of improved sportive individuals. Here, we aimed to knock out the myostatin gene (MSTN), a negative regulator of muscle mass development, using CRISPR/Cas9 and to generate edited embryos for the first time in horses. We nucleofected horse fetal fibroblasts with 1, 2 or 5 µg of 2 different gRNA/Cas9 plasmids targeting the first exon of MSTN. We observed that increasing plasmid concentrations improved mutation efficiency. The average efficiency was 63.6% for gRNA1 (14/22 edited clonal cell lines) and 96.2% for gRNA2 (25/26 edited clonal cell lines). Three clonal cell lines were chosen for embryo generation by somatic cell nuclear transfer: one with a monoallelic edition, one with biallelic heterozygous editions and one with a biallelic homozygous edition, which rendered edited blastocysts in each case. Both MSTN editions and off-targets were analyzed in the embryos. In conclusion, CRISPR/Cas9 proved an efficient method to edit the horse genome in a dose dependent manner with high specificity. Adapting this technology sport advantageous alleles could be generated, and a precision breeding program could be developed.

The influence of the industrial area on the pollution outside its borders: a case study from Quintero and Puchuncavi districts, Chile.

The specific objectives of this study were: (1) to establish background content levels of potentially harmful elements in topsoil of Quintero and Puchuncavi districts; (2) to assess the degree of topsoil pollution and identify the local sources of pollution; and (3) to assess the health risk of metal exposure to population. Data from a Chilean Environmental Ministry report were analyzed. Multivariate analysis suggests that the potentially harmful elements are split into two main groups, which can be explained by controlling geogenic (Ni, V and Zn) and anthropogenic (As, Cd, Cu and Pb) factors. The spatial distribution of As, Cd, Cu and Pb displays a spatial pattern around the Puchuncavi-Ventanas industrial park. The background values determined with MAD (median absolute deviation) method include As 31.6 mg kg-1, Cd 0.7 mg kg-1, Cu 296 mg kg-1, Ni 16.9 mg kg-1, Pb 48.6 mg kg-1, V 199 mg kg-1 and Zn 192 mg kg-1. The pollution indices reported considerable contamination to very high contamination in some soil samples. Hazard quotient values were higher for children than for adults, due to the ingestion and dermal absorption, while the cancer risk index values were in the acceptable range.