Isozygous and selectable marker-free MSTN knockout cloned pigs generated by the combined use of CRISPR/Cas9 and Cre/LoxP

Abstract

Predictable, clean genetic modification (GM) in livestock is important for reliable phenotyping and biosafety. Here we reported the generation of isozygous, functional myostatin (MSTN) knockout cloned pigs free of selectable marker gene (SMG) by CRISPR/Cas9 and Cre/LoxP. CRISPR/Cas9-mediated homologous recombination (HR) was exploited to knock out (KO) one allele of MSTN in pig primary cells. Cre recombinase was then used to excise the SMG with an efficiency of 82.7%. The SMG-free non-EGFP cells were isolated by flow cytometery and immediately used as donor nuclei for nuclear transfer. A total of 685 reconstructed embryos were transferred into three surrogates with one delivering two male live piglets. Molecular testing verified the mono-allelic MSTN KO and SMG deletion in these cloned pigs. Western blots showed approximately 50% decrease in MSTN and concurrent increased expression of myogenic genes in muscle. Histological examination revealed the enhanced myofiber quantity but myofiber size remained unaltered. Ultrasonic detection showed the increased longissimus muscle size and decreased backfat thickness. Precision editing of pig MSTN gene has generated isozygous, SMG-free MSTN KO cloned founders, which guaranteed a reliable route for elite livestock production and a strategy to minimize potential biological risks.

Figure 1. CRISPR/Cas9 mediates efficient and precise HR in both immortal and primary porcine cells.

(A) Cleavage efficiency of targeting sites within the pig MSTN gene. The three selected target sites for homologous recombination (T1, T2 and T3, shown at the top of the panel) were analyzed using the SSA reporter assay. *, significantly different. Error bar, mean ± SD. (B) Strategy for MSTN targeting and SMG deletion. Depicted is i) the schematic outline of the pig MSTN gene, with exons shown in orange and the homology used in the donor DNA in purple (5′ homology arm) and blue (3′ homology arm); ii) schematic map of the donor DNA with homology arms and floxed (LoxP, grey arrow heads) SMG cassette (green rectangle); iii) the correctly targeted locus and iv) the clean targeted locus and the excised SMG following Cre/LoxP-mediated recombination. Primers used for junction and long range PCR are indicated by half arrows. Restriction sites (vertical arrow) and probe (3′ homology arm, blue rectangle with double slash) for Southern blot are shown. (C) Validation of MSTN targeting in PK15 cells. For a correct HR event, a 9898 bp fragment was detected in the correctly targeted locus (KO). A 6869 bp fragment was also detected (WT), indicating that the HR was mono-allelic. NC, negative control using the PK15 genomic DNA. The detected fragments migrated slightly faster than corresponding marker because of the high AT-content (AT% = 66.84%) of the MSTN locus. (D) Junction and long range PCR for detecting MSTN targeting in pig primary cells (T31 cell clone). 5′and 3′junction PCR produced a 962 bp band and a 1503 bp band, respectively. Long range PCR produced a 1737 bp band for WT allele and a 4773 bp band for KO allele (red arrow). M1 and M2 were DL2000 and 1 kb DNA ladders, respectively. H₂O, blank control; wt, wild-type genomic DNA. (E) Sequencing of the junction PCR products. The top and bottom sequences are for 5′and 3′ junctions, respectively. The fluorescence readout is shown for one of the clones, the others being identical. Black and red arrows stand for the native genomic sequences and the homolog sequences, respectively.

Figure 2. Cre-mediated SMG deletion and isolation of marker-free donor cells.

(A) Detection of Cre-mediated SMG deletion by end-point PCR. Primer pair M5F and M3R was used to generate a 396 bp band (clean locus), a 534 bp band (WT allele) and a 3357 bp band (KO allele). Mock: non-electroporated T31 cells; T31: T31 cells electroporated with buffer only and T31/Cre: T31 cells electroporated with Cre mRNA. (B) Efficiency of SMG deletion. Shown are relative SMG copy numbers determined by real-time PCR in T31 cell electroporated with buffer only (T31, black bar) and T31 cell electroporated with Cre mRNA (T31/Cre, green bar). *, significantly different. Error bar, mean ± SD from technical triplicates. (C) Flow cytometery of GFP florescence emitted by wild type porcine embryonic fibroblasts (blank), T31 cells transfected with buffer only (T31) and T31 cells transfected with Cre mRNA (T31/Cre). The results for the different samples are graphically represented as cell numbers (counts) versus GFP average fluorescence (GFP-A). (D) Quantification of GFP-positive cells. The amount of GFP-positive cells was quantified from the cell populations of blank, T31 and T31/Cre samples. The non-GFP fluorescing cells from the Cre mRNA-treated T31 cell population (T31/Cre) in this experiment were sorted by flow cytometery and used for SCNT.

Figure 3. Production and genotyping of SMG-free MSTN KO pigs via SCNT.

(A) Image (200 × magnification) of SCNT embryos reconstructed using wild type cells (WT) and SMG-free MSTN KO cells as nuclei donors. (B) Picture of the two newborn SMG-free MSTN KO piglets produced by SCNT. (C) Confirmation of genotype of the cloned SMG-free MSTN KO piglets by Southern blot. WT: wild-type control; Δ1 and Δ2: SMG-free MSTN KO pigs, respectively. (D) Sequence of the SMG-free MSTN locus from pig Δ1. The black frame highlights the remaining single LoxP motif and the arrows indicate adjacent endogenous sequences homologous to the 5′ and 3′ arms used in the donor DNA.

Figure 4. Molecular and phenotypic analysis of the SMG-free MSTN KO cloned pigs.

(A) Western blot for MSTN and myogenesis-associated proteins. Immunoblots with antibodies specific for the proteins indicated on the left using muscle protein extracts derived from wild type (WT) and the two SMG-free MSTN KO piglets (Δ1, Δ2). Relative quantification of the detected protein levels (WT = 1.0) is shown as bar graphs (WT, black; MSTN KO, green) on the right. MSTN; myoststin; MyoD1: myoblast determination protein 1; MyoG: myogenin; Myf5: myogenic factor 5; GAPDH: glyceraldehyde-3-phosphate dehydrogenase, serving the internal control for equal loading amount. Error bar, mean ± SD. (B) Representative images (100× magnification) of stained muscle sections from wild type (WT) and SMG-free MSTN KO piglets (Δ1, Δ2) to compare quantity and size of myofiber in an identical unit area. (C) Quantification of the number and size of myofibers. The quantity of myofibers is shown as black bars (average of 10 random sections) and the size of the myofibers was measured in arbitrary units (AU) of the cross section areas (CSA) of 100 myofibers (green bars). *, statistically significant. Error bar, mean ± SD. (D) The longissimus muscle size (black bars) and backfat thickness (green bars) of wild type (WT) pigs compared to SMG-free MSTN KO pigs (Δ1, Δ2). Shown are the mean values of measurements at three different spots along the muscle with error bars representing mean ± SD. *, statistically significant. (E) Growth curve of the SMG-free MSTN KO and wild type (WT) pigs from birth to 6-month old. Shown are the average growth curves for the two KO piglets (green) and wild type controls (black; n = 3). *, statistically significant. Error bar, mean ± SD.