Massive crossover suppression by CRISPR–Cas-mediated plant chromosome engineering

Basics of recombination and chromosomal inversions

During meiosis (formation of germ cells), the number of chromosomes is halved, so that as a result male and female germ cells only have a single set of chromosomes. At the beginning of meiosis, when two sets of chromosomes exist, homologue chromosomes pair with each other. This often results in a crossing-over: an exchange of corresponding partial sections of homologous chromosomes of maternal and paternal origin. This process, known as intrachromosomal recombination, only occurs in sexual reproduction. It recombines genetic material and increases the genetic variation in the offspring.

As yet, this process cannot be controlled in either plant or animal breeding. If, for example, a plant has several genes with the desired traits, there is no guarantee that all of them will be passed on to the offspring. Desired genes can be partially separated and recombined by recombination.

Chromosomal inversion is a naturally occurring mechanism that can prevent crossing-over. Such inversions can occur if a chromosome breaks at two sites. The process involves the segment between the two breaks in the chromosome being reinserted in the opposite direction. If an inverted chromosome pairs with the homologue chromosome, a crossover becomes less likely due to the inversion, and the genetic exchange does not occur. The genes within the inversion are therefore inherited en bloc. When a crossover occurs, the resulting gametes are often not viable, as the gene segments are either completely lost or duplicated. Therefore, inversions sometimes greatly reduce the fertility of an organism. This is why especially large inversions are rarely found in nature (Kirkpatrick 2010).

Results of the Rönspies et al. study (2022)

The Rönspies et al. study investigated whether a chromosome in Arabidopsis could be excluded from recombination. For this purpose, a very large inversion was introduced into the chromosome using CRISPR/Cas9. Thus, preventing the crossover of this chromosome with the homologue chromosome so that the genes on the chromosome pass to the offspring in an unchanged combination.

The CRISR/Cas9 inversion introduced by Rönspies et al. covered almost the entire chromosome. Only the ends, i.e. the telomeres, remained in their natural orientation. Examination of the offspring of the plant with the inverted chromosome showed that fertility was, as expected, partially reduced.

In order to test the hypothesis of whether intrachromosomal recombination can be prevented by the CRISPR/Cas9 inversion of the chromosome, the frequencies were examined. In comparison to the control, crossover events were significantly less frequent in plants with CRISPR/Cas9 inversion. If recombination did occur, it was almost exclusively restricted to the regions of the telomeres outside the inverted region. Very rarely, however, complex recombinations also occurred in the inverted regions, resulting in viable germ cells.

Relevance of the results

The results prove that by reconstructing the chromosome using CRISPR/Cas9, it is possible to largely suppress recombination.

However, such inversions do not remain without consequences for the genes located on the chromosome. On the one hand, the inversion does not change the DNA sequence, but DNA sequences are broken off at the beginning and end of the inversion and strung together again. This can result in incomplete genes or new gene products. On the other hand, the position of the genes also changes, which can have a massive influence on the expression of the gene products.

The inversion could not only negatively affect fertility, but also the adaptability of the plant (Roesti et al., 2022). The advantage of suppressing genetic exchange may be accompanied by a simultaneous limitation of adaptation to new ecological conditions. In addition, harmful mutations can accumulate on the inverted segment because they are excluded from the recombination.

This study and other previous studies show that the CRISPR/Cas9 gene scissors can be used to intervene very deeply in the natural recombination and inheritance of genes. For example, coupled genes were separated (Roldan et al., 2017, Schmidt et al., 2020) or the frequency of recombination of inactive, protected genes was increased (Sarno et al., 2017). The approach taken in the above study shows another very powerful application of gene scissors with high technical potential that goes beyond the limits of any previous breeding.

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