Single-Step qPCR and dPCR Detection of Diverse CRISPR-Cas9 Gene Editing Events In Vivo.
Single-Step qPCR and dPCR Detection of Diverse CRISPR-Cas9 Gene Editing Events In Vivo.
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DOI：
10.1534/g3.117.300123
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发表时间：
2017-10-05
期刊：
影响因子：
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通讯作者：
Kaufman BA
中科院分区：
文献类型：
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作者：
Falabella M;Sun L;Barr J;Pena AZ;Kershaw EE;Gingras S;Goncharova EA;Kaufman BA
基于CRISPR-CRISPR相关蛋白9（Cas9）的技术是目前通过重组产生靶向突变或通过非同源末端连接产生indel突变的最灵活手段。在小鼠转基因过程中，重组和插入缺失等位基因通常同时进行。在每只动物中可以形成多个等位基因，从而产生显著的遗传复杂性，这使得CRISPR-Cas9方法和分析变得复杂。目前，还没有快速的方法来测量具有广泛突变敏感性的现场编辑的程度。在这项研究中，我们证明了创始小鼠中靶向CRISPR编辑产生的等位基因多样性。使用这种DNA样品收集，我们验证了用于测量创始小鼠中靶向编辑频率的特异性定量和数字PCR方法（分别为qPCR和dPCR）。我们发现，锁核酸（LNA）探针与内部参考探针（脱落测定）组合提供了编辑速率的准确测量。脱落LNA测定还检测到囊胚中的靶向CRISPR-Cas9基因编辑，其灵敏度与PCR克隆测序相当。最后，我们证明了在qPCR竞争测定中使用的等位基因特异性LNA探针可以准确地检测创始小鼠中的重组突变。总之，我们表明，基于LNA的qPCR和dPCR测定提供了一种快速方法，用于定量体内靶基因组编辑的程度，测试RNA指导和检测重组突变。
Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR-associated protein 9 (Cas9)-based technology is currently the most flexible means to create targeted mutations by recombination or indel mutations by nonhomologous end joining. During mouse transgenesis, recombinant and indel alleles are often pursued simultaneously. Multiple alleles can be formed in each animal to create significant genetic complexity that complicates the CRISPR-Cas9 approach and analysis. Currently, there are no rapid methods to measure the extent of on-site editing with broad mutation sensitivity. In this study, we demonstrate the allelic diversity arising from targeted CRISPR editing in founder mice. Using this DNA sample collection, we validated specific quantitative and digital PCR methods (qPCR and dPCR, respectively) for measuring the frequency of on-target editing in founder mice. We found that locked nucleic acid (LNA) probes combined with an internal reference probe (Drop-Off Assay) provide accurate measurements of editing rates. The Drop-Off LNA Assay also detected on-target CRISPR-Cas9 gene editing in blastocysts with a sensitivity comparable to PCR-clone sequencing. Lastly, we demonstrate that the allele-specific LNA probes used in qPCR competitor assays can accurately detect recombinant mutations in founder mice. In summary, we show that LNA-based qPCR and dPCR assays provide a rapid method for quantifying the extent of on-target genome editing in vivo, testing RNA guides, and detecting recombinant mutations.