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Efficient Generation of Myostatin Knock-Out Sheep Using CRISPR/Cas9 Technology and Microinjection into Zygotes.

Crispo M, Mulet AP, Tesson L, Barrera N, Cuadro F, dos Santos-Neto PC, Nguyen TH, Crénéguy A, Brusselle L, Anegón I, Menchaca A - PLoS ONE (2015)

Bottom Line: When embryo development of CRISPR/Cas9 microinjected zygotes (n = 216) was compared with buffer injected embryos (n = 183) and non microinjected embryos (n = 173), cleavage rate was lower for both microinjected groups (P<0.05) and neither was affected by CRISPR/Cas9 content in the injected medium.Eight showed mutations in both alleles and five of them were homozygous for indels generating out-of frame mutations that resulted in premature stop codons.In conclusion, our results demonstrate that CRISPR/Cas9 system was a very efficient tool to generate gene KO sheep.

View Article: PubMed Central - PubMed

Affiliation: Unidad de Animales Transgénicos y de Experimentación (UATE), Institut Pasteur de Montevideo, Montevideo, Uruguay.

ABSTRACT
While CRISPR/Cas9 technology has proven to be a valuable system to generate gene-targeted modified animals in several species, this tool has been scarcely reported in farm animals. Myostatin is encoded by MSTN gene involved in the inhibition of muscle differentiation and growth. We determined the efficiency of the CRISPR/Cas9 system to edit MSTN in sheep and generate knock-out (KO) animals with the aim to promote muscle development and body growth. We generated CRISPR/Cas9 mRNAs specific for ovine MSTN and microinjected them into the cytoplasm of ovine zygotes. When embryo development of CRISPR/Cas9 microinjected zygotes (n = 216) was compared with buffer injected embryos (n = 183) and non microinjected embryos (n = 173), cleavage rate was lower for both microinjected groups (P<0.05) and neither was affected by CRISPR/Cas9 content in the injected medium. Embryo development to blastocyst was not affected by microinjection and was similar among the experimental groups. From 20 embryos analyzed by Sanger sequencing, ten were mutant (heterozygous or mosaic; 50% efficiency). To obtain live MSTN KO lambs, 53 blastocysts produced after zygote CRISPR/Cas9 microinjection were transferred to 29 recipient females resulting in 65.5% (19/29) of pregnant ewes and 41.5% (22/53) of newborns. From 22 born lambs analyzed by T7EI and Sanger sequencing, ten showed indel mutations at MSTN gene. Eight showed mutations in both alleles and five of them were homozygous for indels generating out-of frame mutations that resulted in premature stop codons. Western blot analysis of homozygous KO founders confirmed the absence of myostatin, showing heavier body weight than wild type counterparts. In conclusion, our results demonstrate that CRISPR/Cas9 system was a very efficient tool to generate gene KO sheep. This technology is quick and easy to perform and less expensive than previous techniques, and can be applied to obtain genetically modified animal models of interest for biomedicine and livestock.

No MeSH data available.


Related in: MedlinePlus

Sequence analysis of lambs’ MSTN exon 1.The same DNAs analyzed in Fig 2 were PCR amplified and the amplicons directly sequenced. In some animals (#40, 47, 49, 50, 56, 57 and 60) DNA from muscle biopsies were PCR amplified and amplicons were cloned into plasmids by TA cloning and electroporated into bacteria, followed by sequencing of 8–10 bacterial clones. A) Depicts for each of the 22 delivered lambs the flanking DNA sequences (in blue) close to the targeted sgRNA sequence (in red) and the PAM sequence (violet); missing nucleotides are represented by spaces, added ones in green and small characters and stop codons are labeled in black. The column Genotype recapitulates the genotype found for each lamb. The column TA indicates the number of bacterial colonies that were sequenced for each allele of the muscle biopsies. The column Translation depicts the aminoacids translated; spaces for the missing ones, in green the ones that are new due to the shift in the coding reading frame and the * represents the stop of the aminoacid sequence due to the premature stop codons. Results are representative of two different PCR amplicons sequencing for all animals. B) A representative sequence electrophoresis, in this case the one of animal #43 which has a biallelic identical deletion of 20 nt.
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pone.0136690.g003: Sequence analysis of lambs’ MSTN exon 1.The same DNAs analyzed in Fig 2 were PCR amplified and the amplicons directly sequenced. In some animals (#40, 47, 49, 50, 56, 57 and 60) DNA from muscle biopsies were PCR amplified and amplicons were cloned into plasmids by TA cloning and electroporated into bacteria, followed by sequencing of 8–10 bacterial clones. A) Depicts for each of the 22 delivered lambs the flanking DNA sequences (in blue) close to the targeted sgRNA sequence (in red) and the PAM sequence (violet); missing nucleotides are represented by spaces, added ones in green and small characters and stop codons are labeled in black. The column Genotype recapitulates the genotype found for each lamb. The column TA indicates the number of bacterial colonies that were sequenced for each allele of the muscle biopsies. The column Translation depicts the aminoacids translated; spaces for the missing ones, in green the ones that are new due to the shift in the coding reading frame and the * represents the stop of the aminoacid sequence due to the premature stop codons. Results are representative of two different PCR amplicons sequencing for all animals. B) A representative sequence electrophoresis, in this case the one of animal #43 which has a biallelic identical deletion of 20 nt.

Mentions: Sequencing of PCR amplicons compassing the targeted MSTN exon 1 sequence showed that ten animals contained mutations; nine identified using capillary electrophoresis and animal #43 which showed an identical deletion of 20 nt in both chromosomes (Fig 3A and 3B), explaining the smaller size of the amplicons in capillary electrophoresis and the negative T7EI assay. The other 12 animals showed only WT alleles. Among the ten mutated animals, eight showed both alleles mutated (#40, 43, 47, 49, 50, 56, 57 and 60) and two only one allele (#44 and 51). These introduced mutations disrupted the coding frame of exon 1 (number of indels different of 3nt or multiples of 3nt) with the generation of premature stop codons in nine mutated animals in at least one of the two alleles. The exception was animal #44 which showed an in-frame shift mutation (Fig 3A). Newly generated stop codons (Fig 3A) are recognized by cells as premature stop codons due to the absence of other normal Cis sequences targeting these mRNAs for degradation through nonsense-mediate decay pathway resulting in the complete absence of the protein [36]. Some animals showed biallelic frameshift mutations introducing premature stop codons (#40, 43, 47, 57 and 60), and are thus homozygous KO animals. Other animals showed one frameshift mutation and another with inframe mutations (#49, 50 and 56) and are thus heterozygous KO animals. Analogously, one animal (#51) showed one allele with a frameshift mutation and a WT allele and thus it is also a heterozygous KO animal. Finally, one animal (#44) had one allele with an inframe mutation and one wild-type allele and thus it is not a KO animal.


Efficient Generation of Myostatin Knock-Out Sheep Using CRISPR/Cas9 Technology and Microinjection into Zygotes.

Crispo M, Mulet AP, Tesson L, Barrera N, Cuadro F, dos Santos-Neto PC, Nguyen TH, Crénéguy A, Brusselle L, Anegón I, Menchaca A - PLoS ONE (2015)

Sequence analysis of lambs’ MSTN exon 1.The same DNAs analyzed in Fig 2 were PCR amplified and the amplicons directly sequenced. In some animals (#40, 47, 49, 50, 56, 57 and 60) DNA from muscle biopsies were PCR amplified and amplicons were cloned into plasmids by TA cloning and electroporated into bacteria, followed by sequencing of 8–10 bacterial clones. A) Depicts for each of the 22 delivered lambs the flanking DNA sequences (in blue) close to the targeted sgRNA sequence (in red) and the PAM sequence (violet); missing nucleotides are represented by spaces, added ones in green and small characters and stop codons are labeled in black. The column Genotype recapitulates the genotype found for each lamb. The column TA indicates the number of bacterial colonies that were sequenced for each allele of the muscle biopsies. The column Translation depicts the aminoacids translated; spaces for the missing ones, in green the ones that are new due to the shift in the coding reading frame and the * represents the stop of the aminoacid sequence due to the premature stop codons. Results are representative of two different PCR amplicons sequencing for all animals. B) A representative sequence electrophoresis, in this case the one of animal #43 which has a biallelic identical deletion of 20 nt.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4549068&req=5

pone.0136690.g003: Sequence analysis of lambs’ MSTN exon 1.The same DNAs analyzed in Fig 2 were PCR amplified and the amplicons directly sequenced. In some animals (#40, 47, 49, 50, 56, 57 and 60) DNA from muscle biopsies were PCR amplified and amplicons were cloned into plasmids by TA cloning and electroporated into bacteria, followed by sequencing of 8–10 bacterial clones. A) Depicts for each of the 22 delivered lambs the flanking DNA sequences (in blue) close to the targeted sgRNA sequence (in red) and the PAM sequence (violet); missing nucleotides are represented by spaces, added ones in green and small characters and stop codons are labeled in black. The column Genotype recapitulates the genotype found for each lamb. The column TA indicates the number of bacterial colonies that were sequenced for each allele of the muscle biopsies. The column Translation depicts the aminoacids translated; spaces for the missing ones, in green the ones that are new due to the shift in the coding reading frame and the * represents the stop of the aminoacid sequence due to the premature stop codons. Results are representative of two different PCR amplicons sequencing for all animals. B) A representative sequence electrophoresis, in this case the one of animal #43 which has a biallelic identical deletion of 20 nt.
Mentions: Sequencing of PCR amplicons compassing the targeted MSTN exon 1 sequence showed that ten animals contained mutations; nine identified using capillary electrophoresis and animal #43 which showed an identical deletion of 20 nt in both chromosomes (Fig 3A and 3B), explaining the smaller size of the amplicons in capillary electrophoresis and the negative T7EI assay. The other 12 animals showed only WT alleles. Among the ten mutated animals, eight showed both alleles mutated (#40, 43, 47, 49, 50, 56, 57 and 60) and two only one allele (#44 and 51). These introduced mutations disrupted the coding frame of exon 1 (number of indels different of 3nt or multiples of 3nt) with the generation of premature stop codons in nine mutated animals in at least one of the two alleles. The exception was animal #44 which showed an in-frame shift mutation (Fig 3A). Newly generated stop codons (Fig 3A) are recognized by cells as premature stop codons due to the absence of other normal Cis sequences targeting these mRNAs for degradation through nonsense-mediate decay pathway resulting in the complete absence of the protein [36]. Some animals showed biallelic frameshift mutations introducing premature stop codons (#40, 43, 47, 57 and 60), and are thus homozygous KO animals. Other animals showed one frameshift mutation and another with inframe mutations (#49, 50 and 56) and are thus heterozygous KO animals. Analogously, one animal (#51) showed one allele with a frameshift mutation and a WT allele and thus it is also a heterozygous KO animal. Finally, one animal (#44) had one allele with an inframe mutation and one wild-type allele and thus it is not a KO animal.

Bottom Line: When embryo development of CRISPR/Cas9 microinjected zygotes (n = 216) was compared with buffer injected embryos (n = 183) and non microinjected embryos (n = 173), cleavage rate was lower for both microinjected groups (P<0.05) and neither was affected by CRISPR/Cas9 content in the injected medium.Eight showed mutations in both alleles and five of them were homozygous for indels generating out-of frame mutations that resulted in premature stop codons.In conclusion, our results demonstrate that CRISPR/Cas9 system was a very efficient tool to generate gene KO sheep.

View Article: PubMed Central - PubMed

Affiliation: Unidad de Animales Transgénicos y de Experimentación (UATE), Institut Pasteur de Montevideo, Montevideo, Uruguay.

ABSTRACT
While CRISPR/Cas9 technology has proven to be a valuable system to generate gene-targeted modified animals in several species, this tool has been scarcely reported in farm animals. Myostatin is encoded by MSTN gene involved in the inhibition of muscle differentiation and growth. We determined the efficiency of the CRISPR/Cas9 system to edit MSTN in sheep and generate knock-out (KO) animals with the aim to promote muscle development and body growth. We generated CRISPR/Cas9 mRNAs specific for ovine MSTN and microinjected them into the cytoplasm of ovine zygotes. When embryo development of CRISPR/Cas9 microinjected zygotes (n = 216) was compared with buffer injected embryos (n = 183) and non microinjected embryos (n = 173), cleavage rate was lower for both microinjected groups (P<0.05) and neither was affected by CRISPR/Cas9 content in the injected medium. Embryo development to blastocyst was not affected by microinjection and was similar among the experimental groups. From 20 embryos analyzed by Sanger sequencing, ten were mutant (heterozygous or mosaic; 50% efficiency). To obtain live MSTN KO lambs, 53 blastocysts produced after zygote CRISPR/Cas9 microinjection were transferred to 29 recipient females resulting in 65.5% (19/29) of pregnant ewes and 41.5% (22/53) of newborns. From 22 born lambs analyzed by T7EI and Sanger sequencing, ten showed indel mutations at MSTN gene. Eight showed mutations in both alleles and five of them were homozygous for indels generating out-of frame mutations that resulted in premature stop codons. Western blot analysis of homozygous KO founders confirmed the absence of myostatin, showing heavier body weight than wild type counterparts. In conclusion, our results demonstrate that CRISPR/Cas9 system was a very efficient tool to generate gene KO sheep. This technology is quick and easy to perform and less expensive than previous techniques, and can be applied to obtain genetically modified animal models of interest for biomedicine and livestock.

No MeSH data available.


Related in: MedlinePlus