Index pcr primers

Off-target analysis was performed as described previously [13 (link)]. The COSMID webtool was used to determine the predicted off-target candidates. The genomic regions flanking potential off-target sites were amplified by two-step PCRs using specific primers (S2 Table) and the Index PCR Primers following the manufacturer’s instructions (Illumina), and subjected to a MiSeq sequencing analysis. Indel or substituted mutations were measured within a 5-bp window around the predicted Cas9 cleavage site in each off-target sites using CRISPResso [68 (link)] to minimize false-positive classification. A small number of amplicons carrying different sequences that were also detected in a WT sample were excluded as sequencing errors.

Genomic DNA was isolated from ear biopsies by boiling in a 50 mM NaOH solution. After neutralization, the genomic regions flanking the gRNA target sequences were amplified by two-step PCR using specific primers and the index PCR primers following the manufacturer’s instructions (Illumina, Hayward, CA, USA) (Supplementary Table S2). After gel purification, the amplicons were subjected to MiSeq sequencing using the MiSeq Reagent Kit v. 2 (250 cycles) (Illumina, San Diego, CA, USA). CRISPResso2^{56 (link)} was used for data analysis. The genotypes of piglets were classified according to the definition of genotypes in embryos described above.
Genomic DNA was isolated from ear, muscle, lung, heart, liver, and kidney by boiling in 50 mM NaOH. After neutralization, the DNA samples were subjected to PCR using specific primers targeting MSTN (Supplementary Table S1). The PCR products were extracted by agarose gel electrophoresis and subjected to Sanger sequencing as described above.

The ear biopsies were collected from piglets under anesthesia by continuous inhalation of 2 to 3% isoflurane. Genomic DNA was isolated from the ear biopsies by boiling in a 50 mM NaOH solution. After neutralization, the genomic regions that flanked the gRNA target sequences were amplified by two-step PCR by specific primers (S1 Table) and the Index PCR Primers following the manufacturer’s instructions (Illumina, Hayward, CA, USA). After gel purification, the amplicons were subjected to MiSeq sequencing using the MiSeq Reagent Kit v. 2 (250 cycles) (Illumina, San Diego, CA, USA). The mutation rates were defined as the ratio of the number of mutant amplicons to the total read number. A small number of amplicons carrying different sequences, that were also detected in WT samples were excluded as sequencing errors. Piglets that carried no WT sequences were classified as having biallelic mutations. Those carrying more than one type of mutation and the WT sequence were classified as mosaics. Piglets that carried only the WT sequence were classified as WT.
For the evaluation of genotype in major organs, genomic DNA isolated from the heart, lung, liver, pancreas, and kidney were amplified by two-step PCR and subjected to MiSeq sequencing as described above.