Detection of nucleotide-specific CRISPR/Cas9 modified alleles using multiplex ligation detection.
Detection of nucleotide-specific CRISPR/Cas9 modified alleles using multiplex ligation detection.
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DOI：
10.1038/srep32048
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发表时间：
2016-08-25
影响因子：
4.6
通讯作者：
Bielas SL
中科院分区：
文献类型：
--
作者：
Kc R;Srivastava A;Wilkowski JM;Richter CE;Shavit JA;Burke DT;Bielas SL
CRISPR/Cas9基因组编辑已经成为在模式生物中创建突变等位基因的强大工具。然而，创建这些突变的精确性为基因分型和菌落管理引入了一系列新的并发症。在许多实验生物体中的传统基因靶向方法掺入外源DNA和/或等位基因特异性序列，其允许基于PCR产物扩增和大小选择的二进制读出的基因分型策略。相比之下，通过Cas9产生的双链DNA断裂的非同源末端连接（NHEJ）修复产生的等位基因不太适合这种策略。在这里，我们描述了一种新的基因分型策略，该策略具有成本效益，序列特异性，并且允许CRISPR/Cas9编辑等位基因的小插入-缺失和单核苷酸变体特征的准确和有效的多重化。我们表明，连接检测反应（LDR）可用于产生的产品，是序列特异性和独特的检测产品的大小和/或荧光标签。该方法的工作独立的模式生物，并将是有用的菌落管理的突变等位基因不同的几个核苷酸在实验动物菌落变得更加普遍。
CRISPR/Cas9 genome-editing has emerged as a powerful tool to create mutant alleles in model organisms. However, the precision with which these mutations are created has introduced a new set of complications for genotyping and colony management. Traditional gene-targeting approaches in many experimental organisms incorporated exogenous DNA and/or allele specific sequence that allow for genotyping strategies based on binary readout of PCR product amplification and size selection. In contrast, alleles created by non-homologous end-joining (NHEJ) repair of double-stranded DNA breaks generated by Cas9 are much less amenable to such strategies. Here we describe a novel genotyping strategy that is cost effective, sequence specific and allows for accurate and efficient multiplexing of small insertion-deletions and single-nucleotide variants characteristic of CRISPR/Cas9 edited alleles. We show that ligation detection reaction (LDR) can be used to generate products that are sequence specific and uniquely detected by product size and/or fluorescent tags. The method works independently of the model organism and will be useful for colony management as mutant alleles differing by a few nucleotides become more prevalent in experimental animal colonies.