Generation and evaluation of Myostatin knock-out rabbits and goats using CRISPR/Cas9 system

Abstract

Myostatin (Mstn) is a conserved negative regulator of skeletal muscle mass in mammals. However, whether precise disruption of Mstn in livestock can be achieved and safely used to improve meat productivity has not been proven. We applied CRISPR/Cas9 system to generate Mstn knock-out (KO) rabbits and goats and then analyzed the changes in their phenotypes to answer this question. We efficiently generated 24 Mstn KO rabbits out of 32 newborn infants after embryo injection with two sgRNAs targeting rabbit Mstn, and found that the Mstn KO rabbits exhibited increased birthweight and a significantly increase in the weight ratios of the quadriceps and biceps muscles to the whole body. Mstn KO also caused high probability of enlarged tongue phenomenon and severe health problems such as stillbirth and early stage death. Using the same method, one out of four goats was generated with edition at Mstn locus. The early stage growth rate of this goat outperformed the control goats. In conclusion, we efficiently generated Mstn KO rabbits and goats using CRISPR/Cas9 technology. However, Mstn KO causes severe health problems and may also have the same effects on other species. This safety issue must be studied further before applied to animal reproduction processes.

Figure 1. The Cas9 system mediated Mstn gene modifications in cultured rabbit embryos.

(a) Schematic diagram of sgRNAs targeting the rabbit Mstn loci. The primer sets labeled as T7_sgR1 and T7_sgR2 were used for T7E1 cleavage assay at the sgR1 and sgR2 target sites, respectively, and primer set LD was used to detect long-range deletions of Mstn in rabbits developed from embryos that were injected with both sgR1 and sgR2. (b,c) Detection of sgR1 and sgR2 mediated cleavage in eight and six rabbit embryos respectively using the T7E1 cleavage assay. Samples with cleavaged bands were marked with asterisks. C, control embryo injected with only Cas9 mRNA; M, DNA marker. (d,e) DNA sequences of the marked samples in (b,c). TA clones from the PCR products were analyzed by DNA sequencing. The PAM sequences and targeting sites are highlighted in red and in blue, the mutated and inserted bases are indicated in lower case. WT, wild type; pm, point mutation; deletions (−), and insertions (+). N/N indicates the positive colonies out of the total colonies sequenced.

Figure 2. The Cas9 system mediated one-step generation of Mstn KO rabbits.

(a) Mstn KO could resulted in enlarged tongues. The red arrows are used to highlight the enlarged tongues of infants #1-1, #11-1 and #16-2. The control rabbit was randomly selected from the 42 naturally born rabbits, the Treated rabbits were generated by embryo injection and transfer. (b,c) Detection of sgR1 and sgR2 mediated cleavage in newborn rabbits #1-#5 and one aborted fetus (AF) using T7E1 cleavage assay. Rabbits #1, #2 and the AF were only injected with sgR2. Rabbits #3-#5 were injected with both sgR1 and sgR2. Samples with cleavage bands were marked with asterisks. (d) Detection of sgR1 and sgR2 mediated long-range deletions in newborn rabbits #3-#5 using PCR. Samples with long-range deletion were marked with an asterisk. (f–g) DNA sequences of marked (*) samples in (b–d) and other samples with multi-peaks in the chromatogram after Sanger sequencing of the sgR1 and sgR2 target sites. TA clones of the PCR products were analyzed by DNA sequencing. The mutated and inserted bases are indicated in lower case. WT, wild type; pm, point mutation; deletions (−), and insertions (+). N/N indicates the positive colonies of the total colonies sequenced. The red triangles indicate the double strands break sites of sgR1 and sgR2.

Figure 3. Evaluation of the effects of Mstn KO on tongue type, health status, birthweight, and muscularity of rabbit fetuses.

(a,b) The ratios of animals with enlarged tongues (a) and early stage death (b) were significantly higher in Mstn KO rabbits than those of the Control and Mstn WT rabbits. Control (n = 42), WT (n = 8) and KO (n = 24) represent rabbits from Control group, Mstn WT group and Mstn KO group. (c–e) Comparison of the weight ratios of tongue (c) quadriceps muscle (d) and biceps muscle (e) to whole body between control rabbits and Mstn KO rabbits. (f) Comparison of the birthweight of the Treated infants with that of the Control infants. (g) Comparison of the birthweight of Control rabbits, Mstn WT rabbits and Mstn KO infants from the Treated group.

Figure 4. Detection of Mstn and Myogenin expression level in skeletal muscle.

(a) Western blotting analysis of Mstn in skeletal muscle of infant rabbits. (b) Quantification of the relative Mstn protein level by gradation analyses. (c) The relative expression of Myogenin in skeletal muscle was determined using qRT-PCR. The 6 Mstn KO infants tested were #1-1, #1-2, #2-3, #2-7, #11-1 and #16-2, and the 3 control rabbits were n1–n3.

Figure 5. The Cas9 system mediated generation of Mstn KO goat.

(a) Schematic diagram of the sgG targeting goat Mstn loci. The PAM sequences and targeting sites are highlighted in red and blue, respectively. The primer set T7_sgG was used for T7E1 cleavage assay at the sgG target site. (b) Detection of sgG:Cas9-mediated cleavage at Mstn loci of the 5 newborn goats by T7E1 cleavage assay. C, control goat; AF, aborted fetus. (c) Photo of the genome edited goat #3. (d) Indel frequency at the Mstn locus in ear genomic DNA. (e) Distribution of the indel length. (f) Distribution of indel frame phase calculated as the length of indels modulus 3. For example, 1-, 4- and 7-base-pair indels are 3N + 1, 2-, 5- and 8-base-pair indels are 3N + 2 and 3-, 6- and 9-base-pair indels are 3N. The pie chart shows the percentage of each class of indel. (g) Representative views of indels in ear genomic DNA of goat #3 using the Integrative Genomics Viewer. The black or purple bars indicate deletions or insertions, respectively. (h) The birthweight (BW0) (upper graph) and 4 month body weight (BW4) (lower graph) of four live newborn goats and control goat herds. Goat #1 (♂), #2 (♂), and #3 (♀) were from a triplet pregnancy and goat #4 (♀) was from a single pregnancy, control goat herds 5 (n = 5, ♂) and 6 (n = 4, ♀) were from multiple pregnancies, and control goat herds 7 (n = 4, ♂) and 8 (n = 4, ♀) were from single pregnancies. T, M, S represent triplet, multiple (triple and double), and single birth types, respectively.