Knockout of the HMG domain of the porcine SRY gene causes sex reversal in gene-edited pigs

Background and objectives of the study
Scientists at the Institute for Farm Animal Genetics at the Friedrich Loeffler Institute, Germany have used the CRISPR/Cas9 to genome edit pigs so that they develop female reproductive organs even though they have a male set of chromosomes. The authors state that their results can be used to avoid piglet castration, which is standard practice in pig breeding, to prevent boar odour in meat. This is routinely done to improve the taste of the meat. Piglet castration without anesthetics has been legally prohibited in Germany since 2021. In addition, such animals could be used to develop a pig animal model with which to study human hereditary diseases such as disorders of sex development, e.g., Swyer syndrome). The Y chromosome, which carries the SRY gene, is decisive for determining sex in pigs and humans. The pig SRY gene is 85% identical to the human SRY gene. So far, studies in rodents (murine species) have mainly been carried out for such animal models, but the murine SRY gene is only 75% like the human one. The study is basic research, but it is application oriented.

Results of the study
Firstly, CRISPR/Cas9 and two gRNAs were microinjected into fertilized pig egg cells. The gRNA(s) serve as a guide for CRISPR/Cas9 to recognize the target sequence(s) in the genome and to introduce a DNA double-strand break there. The use of two gRNAs causes the deletion of a larger area (loss of a DNA-fragment) within the SRY gene. The resulting (genome-edited) embryos were then transferred to recipient sows. Two of them gave birth to a total of twelve piglets, all of which showed female sex characteristics. Three of these piglets are genetically male because they each have both an X and a Y chromosome. They carry the deletion in the SRY gene, which leads to the female gender-specific properties. One of these three piglets was cloned, in other words, to put it simply: “duplicated”. The offspring all had the same male genetic predisposition but, due to genome editing, they have female sex characteristics. The genome-edited piglets showed normal weight gain compared to non-genome-edited females. In addition, up to a certain age, the scientists could not determine any difference between the external and internal sexual organs. From a certain age, however, the genome-edited piglets showed a clear difference in size in terms of internal sexual characteristics. Subsequent investigations showed that the genome-edited animals are sterile. The authors think the reason for the infertility in the missing second X chromosome, which contains few but important genes that are necessary for the fertility of pigs.

Unintended alterations
The study examined the genomes of the three genome-edited piglets with regard to the presence of off-target effects using routine PCR methods. The authors firstly analyzed the genetic material of a reference genome with the help of software, which calculated the possible DNA regions where unintended off-target effects could occur. Off-target effects are unintended changes (mutations) in the genome that can occur in DNA regions similar to the target area. The authors analyzed the first ten calculated off-target areas of the respective gRNA and found no unintended alterations there. During the cytogenetic examination of the chromosomes, however, it was found that one of the piglets had an unintended change of the 7th chromosome (rearrangement of a chromosome arm). The reason for this inversion is unclear. The authors of the study speculate that it is unlikely that the effect is due to the activity of CRISPR/Cas9, since this insertion did not occur in any of the calculated off-target regions. However, this effect did not occur in the controls or the other genome-edited animals. Thus, it cannot be completely ruled out that the rearrangement is due to an unintended activity of CRISPR/Cas9. The consequences of this inversion and the affected DNA sequences were not investigated further. The authors add, that whole genome sequencing (WGS) procedures should be used to examine the genetic material in an unbiased way to detect any unintended changes to DNA caused by CRISPR/Cas9. WGS methods are modern DNA sequencing methods that enable the exact sequence of the bases of the genetic material to be deciphered. These techniques were not used in the study.