Design and specificity of long ssDNA donors for CRISPR-based knock-in

DOI Web Site 被引用文献2件

書誌事項

公開日
2017-08-21
資源種別
preprint
DOI
  • 10.1101/178905
公開者
openRxiv

説明

<jats:p>Update: November 12th, 2019. The conclusions of this pre-print are outdated. See Authors note on page 2. CRISPR/Cas technologies have transformed our ability to manipulate genomes for research and gene-based therapy. In particular, homology-directed repair after genomic cleavage allows for precise modification of genes using exogenous donor sequences as templates. While both single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA) forms of donors have been used as repair templates, a systematic comparison of the performance and specificity of repair using ssDNA versus dsDNA donors is still lacking. Here, we describe an optimized method for the synthesis of long ssDNA templates and demonstrate that ssDNA donors can drive efficient integration of gene-sized reporters in human cell lines. We next define a set of rules to maximize the efficiency of ssDNA-mediated knock-in by optimizing donor design. Finally, by comparing ssDNA donors with equivalent dsDNA sequences (PCR products or plasmids), we demonstrate that ssDNA templates have a unique advantage in terms of repair specificity while dsDNA donors can lead to a high rate of off-target integration. Our results provide a framework for designing high-fidelity CRISPR-based knock-in experiments, in both research and therapeutic settings.</jats:p>

収録刊行物

  • bioRxiv

    bioRxiv 2017-08-21

    openRxiv

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