We determined the specificity of mutations induced by the CRISPR-Cas9 gene-editing system in tobacco (Nicotiana benthamiana) alleles and subsequent genetic stability. For this, we prepared 248 mutant plants using an Agrobacterium-delivered CRISPR-Cas9 system targeting α-1,3-fucosyltransferase 1 (FucT1) and β-1,2-xylosyltransferase1 (XylT1) genes, for which the mutation rates were 22.5% and 25%, respectively, with 20.5% for both loci. Individuals with wild-type (WT) alleles at the NbFucT1 locus in T0 were further segregated into chimeric progeny (37-54%) in the next generation, whereas homozygous T0 mutants tended to produce more (~70%) homozygotes than other bi-allelic and chimeric progenies in the T1 generation. Approximately 81.8% and 77.4% of the homozygous and bi-allelic mutations in T0 generation, respectively, were stably inherited in the next generation, and approximately 50% of the Cas9-free mutants were segregated in T2 generation. One homozygous mutant (Ta 161-1) with a +1 bp insertion in NbFucT1 and a -4 bp deletion in NbXylT1 was found to produce T2 progenies with the same alleles, indicating no activity of the integrated Cas9 irrespective of the insertion or deletion type. Our results provide empirical evidence regarding the genetic inheritance of alleles at CRISPR-targeted loci in tobacco transformants and indicate the potential factors contributing to further mutagenesis.
Keywords: Cas9-free; Nicotiana benthamiana; allele heredity; multi-locus mutation; segregation ratio.