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• W3120908052 abstract "Abstract Validation of CRISPR-Cas9 editing typically explore the immediate vicinity of the gene editing site and distal off-target sequences, which have led to the conclusion that CRISPR-Cas9 editing is very specific. However, an increasing number of studies suggest that on-target unintended editing events like deletions and insertions are relatively frequent but unfortunately often missed in the validation of CRISPR-Cas9 editing. The deletions may be several kilobases-long and only affect one allele. The gold standard in molecular validation of gene editing is direct sequencing of relatively short PCR amplicons. This approach allows the detection of small editing events but fails in detecting large rearrangements, in particular when only one allele is affected. Detection of large rearrangements requires that an extended region is analyzed and the characterization of events may benefit from long-read sequencing. Here we implemented Xdrop™, a new microfluidic technology that allows targeted enrichment of long regions (~ 100 kb) using just a single standard PCR primer set. Sequencing of the enriched CRISPR-Cas9 gene edited region in 4 cell lines on long- and short -read sequencing platforms unravelled unknown and unintended genome editing events. The analysis revealed accidental kb large insertions in 3 of the cell lines, which remained undetected using standard procedures. We also applied the targeted enrichment approach to identify the integration site of a transgene in a mouse line. The results demonstrate the potential of this technology in gene editing validation as well as in more classic transgenics." @default.
• W3120908052 created "2021-01-18" @default.
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• W3120908052 date "2020-05-30" @default.
• W3120908052 modified "2023-10-14" @default.
• W3120908052 title "Verification of CRISPR editing and finding transgenic inserts by Xdrop™ Indirect sequence capture followed by short- and long- read sequencing" @default.
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• W3120908052 doi "https://doi.org/10.1101/2020.05.28.105718" @default.
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