Fig 1.
CRISPR-Cas9 genome editing activity in transfected ovine cells.
A) Exon 1 of the ovine MSTN gene. The sequence recognized by sgRNA is in italic and bold, from nt 110–130, the PAM sequence TGG is underlined and the rest of exonic sequences in smaller font. B) Ovine cells were transfected with pX330-cas9-MSTN plasmid co-expressing Cas9 and sgRNA and three days later cellular DNA was isolated and subjected to T7EI assay for detecting mutation at the targeted MSTN exon 1. The PCR amplifies a band of 634 bp and uncleaved (asterisk) and cleaved (arrows, 255 bp and 379 bp) products are indicated. Numbers below each lane indicate the percentage of targeted indels. NT: non-transduced cells, T: transfected cells, L: 1Kb DNA. The experiment is representative of two replicates performed with similar results.
Table 1.
Cleavage rate on Day 2 and development rate on Day 6 of embryos that were microinjected into cytoplasm with CRISPR/Cas9 RNA system, buffer injection solution, or non-injected embryos (Control group).
Fig 2.
Genotype of lambs born following microinjection of sgRNA for MSTN exon 1 and Cas9.
DNA was extracted from lambs’ skin biopsies one week after birth, with the exception of lambs #43 and #49 that were stillborn and were biopsied 1–2 hours after delivery. A) Results from PCR amplifying 634 bp. Additional larger bands represent formation of heteroduplex DNA double strands with mismatches between the two strands resulting in slower migration during electrophoresis. B) T7EI assay of lambs produced by zygote injection of CRISPR/Cas9 mRNA, indicating numbers of mutant animals with bands of 255, 379 and 634 bp (#40, 43, 44, 47, 49, 50, 51, 56, 57 and 60) and WT animals with only one band of 634 bp (#41, 42, 45, 46, 48, 52, 53, 54, 55, 58, 59 and 61).
Table 2.
Efficiency obtained with CRISPR/Cas9 system injected into cytoplasm of ovine zygotes to produce MSTN mutant lambs.
Data obtained from 53 embryos transferred into 29 recipient ewes.
Fig 3.
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.
Fig 4.
Muscle biopsies from two representative animals, one homozygous (homoz.) for a frameshift mutation and another heterozygous (heteroz.) for a frameshift mutation and a WT copy in the second allele (# 43 and 44, respectively, the numbers of animals correspond to those of Fig 3) analyzed by western blot using an anti-MSTN monoclonal antibody. After stripping of the anti-MSTN antibody from the membrane, an anti-GAPDH was used as loading control. One representative western blot experiment out of three performed with the same results.
Fig 5.
Body weight in lambs produced by CRISPR/Cas9 system.
Significant diference (P<0.05) from 15 to 60 days after birth was found among homozygous KO and wild-type lambs.
Fig 6.
Phenotype of lambs 30 days after birth.
The lamb at the top is wild type (#48), the lamb at the bottom is a homozygous KO (#47). Note the differences in muscle mass from the posterior limbs and loin from the homozygous KO lamb when compared to wild-type. At the time of this picture, with 30 days old the body weight was 8.750 (#48) and 11.150 kg (#47).