Genetics Research LLC, Waltham, Massachusetts, United States of America.
PLoS One. 2020 Dec 23;15(12):e0243781. doi: 10.1371/journal.pone.0243781. eCollection 2020.
The programmable sequence specificity of CRISPR has found uses in gene editing and diagnostics. This manuscript describes an additional application of CRISPR through a family of novel DNA enrichment technologies. CAMP (CRISPR Associated Multiplexed PCR) and cCAMP (chimeric CRISPR Associated Multiplexed PCR) utilize the sequence specificity of the Cas9/sgRNA complex to target loci for the ligation of a universal adapter that is used for subsequent amplification. cTRACE (chimeric Targeting Rare Alleles with CRISPR-based Enrichment) also applies this method to use Cas9/sgRNA to target loci for the addition of universal adapters, however it has an additional selection for specific mutations through the use of an allele-specific primer. These three methods can produce multiplex PCR that significantly reduces the optimization required for every target. The methods are also not specific to any downstream analytical platform. We additionally will present a mutation specific enrichment technology that is non-amplification based and leaves the DNA in its native state: TRACE (Targeting Rare Alleles with CRISPR-based Enrichment). TRACE utilizes the Cas9/sgRNA complex to sterically protect the ends of targeted sequences from exonuclease activity which digests both the normal variant as well as any off-target sequences.
CRISPR 的可编程序列特异性已在基因编辑和诊断中得到应用。本文通过一系列新型 DNA 富集技术描述了 CRISPR 的另一种应用。CAMP（CRISPR 相关的多重 PCR）和 cCAMP（嵌合 CRISPR 相关的多重 PCR）利用 Cas9/sgRNA 复合物的序列特异性靶向靶标，将通用接头连接到靶标，随后进行扩增。cTRACE（基于 CRISPR 的靶向稀有等位基因的嵌合）也应用该方法将 Cas9/sgRNA 靶向靶标，在靶标中添加通用接头，但它通过使用等位基因特异性引物，对特定突变进行额外选择。这三种方法都可以产生多重 PCR，大大减少了每个靶标所需的优化。这些方法也不受任何下游分析平台的限制。我们还将介绍一种基于 CRISPR 的非扩增的突变特异性富集技术，该技术可以保持 DNA 的原始状态：TRACE（基于 CRISPR 的靶向稀有等位基因的富集）。TRACE 利用 Cas9/sgRNA 复合物来阻止靶向序列末端的核酸外切酶活性，该活性会消化正常变体以及任何脱靶序列。
