CRISPR-Cas9-mediated genome editing in tetraploid alfalfa: data on efficiency, timing, and transmission to the sexual and clonal progeny

Genome editing in autopolyploid plants has the potential to modify all the alleles of a gene of interest at the same time. We attempted to edit the alfalfa glutamate 1-semialdehyde aminotransferase (GSA) gene by CRISPR-Cas9, using a gRNA targeting the second intron and a donor DNA introducing a point mutation for gabaculine resistance. We determined that three GSA copies were present in our lab genotype, and one in three was a previously undetected variant allele differing at the target site, which could not be cut by Cas9. Precise, homology-directed repair was not observed, but five in fourteen transgenic plants carried −1, −3, −4 or −5 deletions at the target site. In three of them, only one of the two standard alleles was mutated. In one event, only the variant allele and a −5 deletion allele were present, demonstrating complete editing of the standard allele. Several rare deletion alleles were detected through deep sequencing, indicating a limited, continued activity of the DSB machinery after in vitro regeneration. No new mutated alleles emerged from sexual reproduction. Interestingly, some clonal progenies from an edited plant presented deletion allele frequencies different from the original plant, likely due to somatic segregation of chimeric mutations. This can possibly be exploited to generate new edited genotypes through clonal reproduction.