Department of Horticulture, Chungnam National University, Daejeon 34134, Korea.
Department of Smart Agriculture Systems, Chungnam National University, Daejeon 34134, Korea.
Int J Mol Sci. 2022 Feb 23;23(5):2450. doi: 10.3390/ijms23052450.
We determined the specificity of mutations induced by the CRISPR-Cas9 gene-editing system in tobacco () alleles and subsequent genetic stability. For this, we prepared 248 mutant plants using an -delivered CRISPR-Cas9 system targeting () and () 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 locus in T were further segregated into chimeric progeny (37-54%) in the next generation, whereas homozygous T mutants tended to produce more (~70%) homozygotes than other bi-allelic and chimeric progenies in the T generation. Approximately 81.8% and 77.4% of the homozygous and bi-allelic mutations in T generation, respectively, were stably inherited in the next generation, and approximately 50% of the Cas9-free mutants were segregated in T generation. One homozygous mutant (Ta 161-1) with a +1 bp insertion in and a -4 bp deletion in was found to produce T 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.
我们确定了 CRISPR-Cas9 基因编辑系统在烟草 () 等位基因中诱导的突变的特异性，以及随后的遗传稳定性。为此，我们使用靶向 () 和 () 基因的 - 递送 CRISPR-Cas9 系统制备了 248 株突变体植物，突变率分别为 22.5%和 25%，两个基因座的突变率均为 20.5%。在 T 中 位点具有野生型（WT）等位基因的个体在下一代中进一步分离为嵌合体后代（37-54%），而纯合 T 突变体倾向于产生比其他双等位基因和嵌合体后代更多的纯合子（~70%）。T 代中约 81.8%和 77.4%的纯合和双等位基因突变分别在下一代中稳定遗传，T 代中约 50%的无 Cas9 突变体被分离。在 T 代中发现一个在 中插入 +1 bp，在 中缺失 -4 bp 的纯合突变体（Ta 161-1）产生具有相同等位基因的 T 后代，表明整合的 Cas9 没有活性，无论插入或缺失类型如何。我们的结果提供了有关烟草转化体中 CRISPR 靶向基因座等位基因遗传的经验证据，并指出了导致进一步突变的潜在因素。
