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

Abstract

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 Arabidopsis thaliana 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.

Crop Improvement: Comparison of Transgenesis and Gene Editing

“…The integration occurs according to the NHEJ or HDR mechanism, depending on the presence of homology regions surrounding the inserted DNA. Both mechanisms can lead to mutations and rearrangements at the ends of the inserted DNA region [110]. Moreover, while in experiments on animal cells, there are examples of highly efficient integration through the NHEJ mechanism [111], in the case of plants, the efficiency of integration through this mechanism is usually not so high [112].…”

Clustered regularly interspaced short palindromic repeats/CRISPR-associated protein and hairy roots: a perfect match for gene functional analysis and crop improvement

“…While CRISPR/Cas9-targeted mutagenesis have been used in many plants, including legumes ( Table 1 ), HR-mediated targeted insertion remains a big challenge. This is probably because it operates only at the S and G2 phases of the cell cycle 20 , 28 , 29 , but also due to the difficulty of delivering template DNAs to plant cells surrounded by semirigid cell walls. Nonetheless, successful examples of HR (gene targeting) in different crop species have been achieved 6 , 30 .…”

Physiological and Transcriptomic Analyses Reveal the Effects of Carbon-Ion Beam on Taraxacum kok-saghyz Rodin Adventitious Buds

“…HR is a high-fidelity repair pathway that occurs mainly during the S and G2 phases of the cell cycle and often viewed as a high-fidelity pathway [50,51]. However, some studies also indicated that HR caused structural variation such as deletions and duplications [52,53]. NHEJ repair is an error-prone repair pathway that does not depend on the homologous template, and its repair process is not restricted to a certain stage of the cell cycle, often introducing small insertions and deletions in the genome [54,55].…”