Introduction

Genome editing has played a powerful role in various fields. In the same line with other fields, in livestock, genome editing tools such as ZFN, TALENs, and CRISPR/Cas9, has been applied to disease resistance, climate response, allergy free and increasing productivity [1,2,3,4,5,1) and in MSTN, where its phenoty** can be easily detected, it began to be observed (Fig. 3). Other phenoty** will be analyzed later because BLG should be analyzed after pregnancy and for PRNP, its test should be evaluated in the continuously follow up.

As summarized in Fig. 2, there was a unique fact in the genoty** analysis. In previous study, the MSTN mutated cattle showed only −12 bps [1]. However, in this study, although another gene delivery was applied into zygotes via RNP, −12 bps genoty** was dominantly identified as well, and new genoty** (1, 25 bps insertion and 3 bps deletion) was observed. In more details, no more than 2 genoty** was identified in calves with MSTN mutations (Fig. 2). However, three genoty** were observed in PRNP locus, and 3 or more mutations were observed in BLG except for one mutated calf. According to our results so far, −12 bp is dominantly observed in MSTN knockout cattle through microinjection or electroporation on IVF embryos. In the future, we will continue to monitor the results of knockout genoty** in vivo results through various gene loci. And we found very low percentage of off-target mutation in #5 (Additional file 3). After the offspring become at over than 12 months old, the genoty** and off-target analysis in their germ cells will be followed.

One interesting point is about double knockout event. In dairy breed calves, in which MSTN and BLG were targeted, there were high double knockout efficiency in randomly selected single cell colony analysis. However, in beef breed MSTN/PRNP KO calf, double knockout event in the offspring was very low while its event was comparable with that of MSTN/BLG in blastocyst stage. Because there is only one offspring with MSTN/PRNP KO, it is difficult to determine whether this occurrence was caused by specific target gene locus or breed/individual predisposition. We intend to conduct relevant research in the near future.

Recently, a genome edited organism without any recombinant DNA integration can be classified as non-GMO in many countries [9]. For instance, in Japan, MSTN edited fish is approved to be edible as a food chain [21] and in USA, low-risk determination was made for marketing genome-edited cattle [22]. In the case of animals, safety analysis is not necessary, and their health can be considered an indicator of animal product safety [23]. We were able to find no special complications or significant changes in blood analysis when we assessed the health of animals born as double knockouts, indicating that they are currently growing healthily. As a result, it is expected that the safety of meat and milk from these sources will be unaffected in the future.

Conclusions

In conclusion, these data demonstrated that genome editing on bovine embryos via electroporation of RNP could be effectively applied and proved. Finally, beef cattle with MSTN and PRNP mutation and dairy cattle with MSTN and BLG mutation have been born and they will be valuable resources for future precise breeding.