CRISPR/Cas9-Mediated Targeted DNA Integration: Rearrangements at the Junction of Plant and Plasmid DNA

Targeted DNA integration into known locations in the genome has potential advantages over the random insertional events typically achieved using conventional means of genetic modification. We studied the presence and extent of DNA rearrangements at the junction of plant and transgenic DNA in five li...

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Published in:International journal of molecular sciences 2022-08, Vol.23 (15), p.8636
Main Authors: Permyakova, Natalya V, Marenkova, Tatyana V, Belavin, Pavel A, Zagorskaya, Alla A, Sidorchuk, Yuriy V, Deineko, Elena V
Format: Article
Language:English
Subjects:
Arabidopsis
Cell lines
CRISPR
CRISPR-Cas Systems - genetics
Deoxyribonucleic acid
DNA
Flanks
Gene Editing
Gene Rearrangement
Gene therapy
Genes
Genetic engineering
Genetic modification
Genomes
HDR
Histone H3
Histones
Homology
Insertion
knock-in
Mutation
NHEJ
Plants - genetics
Plasmids
Transgenic plants
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Summary:Targeted DNA integration into known locations in the genome has potential advantages over the random insertional events typically achieved using conventional means of genetic modification. We studied the presence and extent of DNA rearrangements at the junction of plant and transgenic DNA in five lines of suspension cells carrying a site-specific integration of target genes. Two types of templates were used to obtain knock-ins, differing in the presence or absence of flanking DNA homologous to the target site in the genome. For the targeted insertion, we selected the region of the histone H3.3 gene with a very high constitutive level of expression. Our studies showed that all five obtained knock-in cell lines have rearrangements at the borders of the integrated sequence. Significant rearrangements, about 100 or more bp from the side of the right flank, were found in all five plant lines. Reorganizations from the left flank at more than 17 bp were found in three out of five lines. The fact that rearrangements were detected for both variants of the knock-in template (with and without flanks) indicates that the presence of flanks does not affect the occurrence of mutations.
ISSN:1422-0067
1661-6596
1422-0067
DOI:10.3390/ijms23158636