Background CRISPR/Cas9-based genome-editing systems have been used to efficiently engineer livestock species with precise genetic alterations intended for biomedical and agricultural applications. Previously, we have successfully generated gene-edited sheep and goats via one-cell-stage embryonic microinjection of a Cas9 mRNA and single-guide RNAs (sgRNAs) mixture. However, most gene-edited animals produced using this approach were heterozygotes. Additionally, non-homozygous gene-editing outcomes may not fully generate the desired phenotype in an efficient manner. Results We report the optimization of a Cas9 mRNA-sgRNA delivery system to efficiently generate homozygous myostatin (MSTN) knockout sheep for improved growth and meat production. Firstly, an sgRNA selection software (sgRNAcas9) was used to preliminarily screen for highly efficient sgRNAs. Ten sgRNAs targeting the MSTN gene were selected and validated in vitro using sheep fibroblast cells. Four out of ten sgRNAs (two in exon 1 and two in exon 2) showed a targeting efficiency > 50%. To determine the optimal CRISPR/Cas9 microinjection concentration, four levels of Cas9 mRNA and three levels of sgRNAs in mixtures were injected into sheep embryos. Microinjection of 100 ng/μL Cas9 mRNA and 200 ng/μL sgRNAs resulted in the most improved targeting efficiency. Additionally, using both the highly efficient sgRNAs and the optimal microinjection concentration, MSTN-knockout sheep were generated with approximately 50% targeting efficiency, reaching a homozygous knockout efficiency of 25%. Growth rate and meat quality of MSTN-edited lambs were also investigated. MSTN-knockout lambs exhibited increased body weight and average daily gain. Moreover, pH, drip loss, intramuscular fat, crude protein, and shear force of gluteal muscles of MSTN-knockout lambs did not show changes compared to the wild-type lambs. Conclusions This study highlights the importance of in vitro evaluation for the optimization of sgRNAs and microinjection dosage of gene editing reagents. This approach enabled efficient engineering of homozygous knockout sheep. Additionally, this study confirms that MSTN-knockout lambs does not negatively impact meat quality, thus supporting the adoption of gene editing as tool to improve productivity of farm animals.
Introduction: Intact functional pancreatic islets can be successfully isolated from various domestic pig breeds, including some minipig strains. Nevertheless, the technically demanding pig islet isolation procedure needs further improvements. Few efforts have been made to determine the underlying mechanisms for the greatly varying islet yields in many domestic pig breeds. This problem also relates to Landrace Pigs that are often used as donor animals to insert different human genes into the pig genome in order to overcome the various immunological reactions in response to transplantation of a porcine xenograft. Methods: To obtain a clearer picture on the efficiency of islet yields, we evaluated the number of islets, their morphology and intensity of insulin staining in pancreata of various brain‐dead pigs of crossbred and pure bred strains. We performed histochemistry with frozen tissue sections and anti‐pig‐insulin antibody and applied an evaluation procedure, based upon >1.600 microscopically screened pancreata, >800 isolations, and sub‐sequent functional in vivo and in vitro assays. Results: In first series 40 pancreata (tail end of the splenic lobe) of both sexes of the following donor animals were investigated: Piétrain [PI] × German Landrace Pig [GL] (n = 5), German Pure Bred Pig [GPB] x GL (n = 3), PI x GL/GPB (n = 9), GL (n = 12), GPB (n = 3), PI (n = 7), and Duroc (n = 1). Pigs were 6‐7 months old, and were fed and kept under identical conditions. Only three of 40 pancreata (7.5%) displayed islets of sufficient quality for subsequent isolation [data not shown], i.e. sufficient numbers of mostly rounded islets of ≥200 μm in diameter, displaying intensive and evenly distributed insulin staining. Each of the 3 “good” donor organs belonged to a different pig breed. This test was repeated a second time, using 50 pancreata from brain‐dead pigs (both sexes) collected in a Bavarian pig breeding institution: PI × GL (n = 16), PI × GL/GPB (n = 16), GL × GPB (n = 5), GL (n = 12), GPB (n = 2), PI (n = 1). Age, keeping and feeding conditions were identical with conditions of the first trial. Overall, four of 50 pancreata (8%) were determined to be suitable for subsequent islet isolation [not shown here], according to our microscopic evaluation criteria. Again, those four “good” pancreata belonged to four different pig breeds, i.e., a correlation between a particular pig breed and “good” islets could not be detected. In both tests, pancreata that displayed islets with large insulin‐free areas, pancreata with insufficient islet numbers, and islets that displayed very weak insulin staining, as compared to positive control islets, were determined as being unsuitable for islet isolation. Conclusions: Only 7.5–8% of domestic pig pancreata (n = 90) appear to be suitable for islet isolation; in previous years this number was 35–50%. The reasons for this continuous decline in suitable donor organs are presently unknown. None of the crossbreeds and pure breeds tested here displayed a genetic background that correlated with...
Hyperacute rejection after porcine-to-human xenotransplantation is caused by binding of preformed human antibodies against Gal-epitopes on the surface of porcine cells. Organs from Gal-negative pigs have shown prolonged survival after transplantation into baboons. Knocking out a gene by conventional gene targeting frequency is extremely inefficient (homologous recombination = 0.0001 to 0.001%; Denning et al. 2001). Recent publications in rats (Geurts et al. 2009) show that the gene knockout via zinc finger nuclease (ZFN)-driven nonhomologous end joining (NHEJ) can be enhanced 10 000-fold over conventional approaches, making it feasible to generate a biallelic gene knockout with one ZFN application. Here, we used ZFN technology to generate porcine cells that carry a ZFN-mediated knockout of the Gal gene to use these cells as donor cells in somatic cell nuclear transfer (SCNT) to obtain live offspring. One primary porcine fetal fibroblast cell line was transfected by electroporation (n = 6) with a pair of ZFN plasmids designed to target the DNA sequence encoding the catalytic domain located in exon 9 of the α1,3-gal locus. Transfected cells were incubated (7 days at a combination of 30°C and 37°C) and analysed for Gal expression by fluorescence activated cell sorting (FACS) using fluorescein isothiocyanate (FITC)-conjugated isolectin-B4. On average, 1.4% (± 0.3%; n = 6) of the cells were free of Gal epitopes, indicating a biallelic knockout. DNA mutation detection analysis (Cel-I assay) of cell cultures gave a mean frequency of 3.5% NHEJ (± 1.3%; n = 6) giving the fraction of mutant alleles within the cell population. One cell line with 1% Gal-negative cells was sorted by a magnetic Dynabead-based separation method to select for Gal-negative cells (Fujimura et al. 2008). Because of the limited amount of Gal-negative cells within the cell population, we chose to select the cells with magnetic beads. This method is gentler to the cells and leads to a higher plating efficiency after sorting compared with FACS. The sorted cells could be easily expanded and will serve as donor cells in SCNT to show the feasibility of generating knockout pigs via ZFN-mediated gene knockout. This study demonstrates that ZFN technology is an applicable tool to produce genetically modified porcine cells for use as donors in SCNT and to speed the creation of pig models for xenotransplantation and human diseases.
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