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