Pmd20

Genomic DNAs were prepared from blastocysts or ear biopsies by boiling them in 50 mM NaOH solution. After neutralization, the genomic regions flanking the sgRNA target sequences were amplified by PCR using the following specific primers: FGF10, 5′-CCATCCCATTTGATCTGCTT-3′ (forward) and 5′-CTTCAACTGGCAGCACAATG-3′ (reverse); MSTN, 5′-ATGCAAAAACTGCAAATCTATG-3′ (forward) and 5′-TGTAGGCATGGTAATGATCG-3′ (reverse). The PCR products were cloned into the pMD20 (Takara Bio) plasmid. More than 12 plasmids were isolated per blastocyst or biopsy, and the targeted genomic regions were sequenced. Sequencing was performed using the BigDye Terminator Cycle Sequencing Kit version 3.1 (Thermo Fisher Scientific) and the ABI 3500 Genetic Analyzer (Applied Biosystems).

Bacterial loads were estimated with quantitative real-time PCR, targeting the V3-V4 region of 16S rRNA gene with the following primers: 5′-ACTCCTACGGGAGGCAGCAGT-3′ (forward) and 5′-ATTACCGCGGCTGCTGGC-3′ (reverse) [70 (link)]. Normalization was done relative to the actin gene of the host, using the following primers for A. mellifera: 5′-TGCCAACACTGTCCTTTCTG-3′ (forward) and 5′-AGAATTGACCCACCAATCCA-3′ (reverse). For A. cerana, the reverse primer was 5′-AGAATTGATCCACCAATCCA-3′. Standards were prepared as also described in [93 (link)]. The target sequence was cloned into plasmid vector pMD-20 (TaKaRa). The insertion of the target sequence was confirmed by sequencing, and the insert was amplified by PCR and purified. The copy number of the PCR product was calculated, serially diluted and used as standard. qPCR reactions were performed in triplicates in a total volume of 10 μL, containing 5 μL of 2 x TB Green premix Ex TaqII, 0.2 μL ROX reference dye II, 0.2 μM of each primer and 1 μL of 100x-diluted extracted DNA, on a QuantStudio 3 instrument (Applied Biosystems). The thermal cycling conditions were as follows: denaturation at 95°C for 30 s, followed by 40 amplification cycles at 95°C for 5 s, and 60°C for 1 min (actin) or 30 s (16S rRNA).