Abstract
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-xylosyltransferase 1 (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.
Highlights
To characterize the pattern of transmission of CRISPR-Cas9-mediated mutations to the T1 generation, we examined the genotypes of the two target regions in selected linethe T1 generation, we examined the genotypes of the two target regions in selected lineages ages of T0 transgenic plants with confirmed mutagenesis
We further investigated the inheritance and stability of mutations in the T2 generation progeny derived from the T1 generation Ta161-1 line, which had inherited mutations occurring in each of the NbFucT1 and NbXylT1 target sites in the homozygous form and retained Cas9
Genetic tendencies have been reported in tomatoes, Arabidopsis, rice, and potatoes [12,13,23–26]
Summary
Breeding strategies are continually being modified in response to changing climates and for the development of crops with superior traits [1]. Compared with other gene-editing technologies, the CRISPR-Cas system has advantages such as the efficiency of target site design, potential for simultaneous multiple editing, and simplicity that does not require complex protein engineering [9] Given these advantages, this technology has been used to modify a wide range of plants including rice, corn, wheat, soybean, barley, tomato, petunia, and lettuce [5,6,10]. To ensure the development of transformant tobacco in which the acquired useful traits are stably maintained through multiple generations, it is necessary to provide information on the genetic pattern, efficiency, and specificity of CRISPR/Cas-induced gene mutations in different cases. 2022, 23, 2450 the α-1,3-fucosyltransferase 1 (NbFucT1) and β-1,2-xylosyltransferase 1 (NbXylT1) genes to evaluate gene editing using the CRISPR-Cas system in tobacco and assessed how these modifications are inherited and segregated in subsequent generations. Pattern in the generation (primarily T1 , and in some cases, T2 )
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