CRISPR/Cas12a mediated knock-in of the Polled Celtic variant to produce a polled genotype in dairy cattle

The aim of the study is to introduce polledness (hornlessness) into dairy cows with the help of the CRISPR/Cas gene scissors. Polledness occurs naturally in cattle breeds such as Angus, which are used in beef cattle breeding, but much less often in dairy cows.
In the study, the scientists used CRISPR/Cas12a, a variant of the “classic” CRISPR/Cas9 gene scissors. Cas12a differs from Cas9, amongst other things, in a specific cutting pattern that the genetic scissors leave behind in the genome. This is intended to promote the integration of DNA templates into the genome of the target organism.
The scientists took some skin cells for cloning from the ear of a Holstein-Friesian cattle, a breed that is often used in milk production. They cultivated these cells in a cell culture and introduced the gene scissors into the cells together with a guide RNA, which determines the target region in the cattle’s genome, and a DNA template for polledness. A total of 70 positive clones were produced in which the additional piece of DNA was inserted into the genome to convey polledness. The nuclei of the altered cells were then injected into previously enucleated (i.e. emptied of the nucleus) egg cells, which are then supposed to develop into embryos. A total of nine embryos were transferred to surrogate cows. Three of the embryos did not induce pregnancy, but died directly in the uterus. Four of the cows suffered serious complications in the course of their pregnancy, and they lost their calves. Another calf was killed prematurely for experimental purposes. Only one calf was born alive by caesarean section, but died the same day. Although it was polled, it had malformations in several organs. The calf also had an increased body weight. The causes of the serious damage to health were not examined in more detail. It is likely that the cloning process played a major role in the undesired outcome of the experiments as cloning is known to result in birth defects.
The study examines the genome of the genome-edited calf only to a limited extent in regard to unintended changes of the genome: at three regions of the genome, so-called PCR methods were used to search for off-target effects. Off-target effects are unwanted changes that can be caused by the genetic scissors in parts of the genome that are very similar to the target sequence. No off-target effects were found in the three areas examined. However, the rest of the genome was not investigated.
In addition, the scientists examined the genome by applying further PCR methods for unintentionally integrated DNA fragments. This results show how limited the informative value of such a biased detection methods is: the scientists cannot completely rule out that there is an additional antibiotic resistance in the genome of the calf, which was used for the work in the laboratory and should no longer be present in the genome of the calf.
In addition, the scientists cannot clearly prove with the PCR method whether the integrated piece of DNA that mediates the polledness has been integrated into the genome of the calf once or several times.
Only with a genome-wide analysis of the genome of the calf using whole genome sequencing methods the scientists would have been able to provide meaningful results on these unintended changes.