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Verification of CRISPR editing and finding transgenic inserts by Xdrop Indirect sequence capture followed by short- and long- read sequencing
bioRxiv - Genomics Pub Date : 2021-01-07 , DOI: 10.1101/2020.05.28.105718
Blondal Thorarinn , Gamba Cristina , Camille M. Johnston , Eva M. Riising , Marie J. Mikkelsen , Mouritzen Peter

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.

中文翻译:

通过Xdrop间接序列捕获以及短读和长读测序验证CRISPR编辑和发现转基因插入物

CRISPR-Cas9编辑的验证通常探索基因编辑位点和远端脱靶序列的紧邻区域,从而得出结论,即CRISPR-Cas9编辑非常特异。然而,越来越多的研究表明,在删除CRISPR-Cas9编辑时,诸如删除和插入之类的针对性意外编辑事件相对频繁,但不幸的是经常错过。缺失可能长达几千个碱基,并且仅影响一个等位基因。基因编辑的分子验证中的金标准是对较短的PCR扩增子进行直接测序。这种方法允许检测较小的编辑事件,但无法检测较大的重排,特别是在仅影响一个等位基因的情况下。检测大的重排需要分析扩展区域,事件的表征可能会受益于长时间阅读的测序。在这里,我们实施了Xdrop,这是一种新的微流技术,仅使用一个标准的PCR引物组就可以靶向富集长区域(〜100 kb)。在长读和短读测序平台上的4个细胞系中富集的CRISPR-Cas9基因编辑区的测序未发现未知和意外的基因组编辑事件。分析显示在3个细胞系中意外kb大插入,使用标准程序仍未检测到。我们还应用了靶向富集方法来鉴定转基因在小鼠品系中的整合位点。结果证明了该技术在基因编辑验证以及更经典的转基因中的潜力。
更新日期:2021-01-07
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