Hodgens Charles, Nimchuk Zachary L, Kieber Joseph J
Curriculum in Genetics and Molecular Biology, University of North Carolina, Chapel Hill, North Carolina, United States of America.
Department of Biology, University of North Carolina, Chapel Hill, North Carolina, United States of America.
PLoS One. 2017 Nov 15;12(11):e0188406. doi: 10.1371/journal.pone.0188406. eCollection 2017.
Genetic manipulation of organisms using CRISPR/Cas9 technology generally produces small insertions/deletions (indels) that can be difficult to detect. Here, we describe a technique to easily and rapidly identify such indels. Sequence-identified mutations that alter a restriction enzyme recognition site can be readily distinguished from wild-type alleles using a cleaved amplified polymorphic sequence (CAPS) technique. If a restriction site is created or altered by the mutation such that only one allele contains the restriction site, a polymerase chain reaction (PCR) followed by a restriction digest can be used to distinguish the two alleles. However, in the case of most CRISPR-induced alleles, no such restriction sites are present in the target sequences. In this case, a derived CAPS (dCAPS) approach can be used in which mismatches are purposefully introduced in the oligonucleotide primers to create a restriction site in one, but not both, of the amplified templates. Web-based tools exist to aid dCAPS primer design, but when supplied sequences that include indels, the current tools often fail to suggest appropriate primers. Here, we report the development of a Python-based, species-agnostic web tool, called indCAPS, suitable for the design of PCR primers used in dCAPS assays that is compatible with indels. This tool should have wide utility for screening editing events following CRISPR/Cas9 mutagenesis as well as for identifying specific editing events in a pool of CRISPR-mediated mutagenesis events. This tool was field-tested in a CRISPR mutagenesis experiment targeting a cytokinin receptor (AHK3) in Arabidopsis thaliana. The tool suggested primers that successfully distinguished between wild-type and edited alleles of a target locus and facilitated the isolation of two novel ahk3 null alleles. Users can access indCAPS and design PCR primers to employ dCAPS to identify CRISPR/Cas9 alleles at http://indcaps.kieber.cloudapps.unc.edu/.
使用CRISPR/Cas9技术对生物体进行基因操作通常会产生难以检测的小插入/缺失（indels）。在此，我们描述了一种轻松快速识别此类indels的技术。通过切割扩增多态性序列（CAPS）技术，可以很容易地将改变限制性内切酶识别位点的序列鉴定突变与野生型等位基因区分开来。如果突变产生或改变了一个限制性位点，使得只有一个等位基因含有该限制性位点，那么可以通过聚合酶链反应（PCR）随后进行限制性消化来区分这两个等位基因。然而，在大多数CRISPR诱导的等位基因情况下，目标序列中不存在这样的限制性位点。在这种情况下，可以使用衍生CAPS（dCAPS）方法，即在寡核苷酸引物中有目的地引入错配以在一个而非两个扩增模板中创建一个限制性位点。存在基于网络的工具来辅助dCAPS引物设计，但当提供包含indels的序列时，当前工具常常无法给出合适的引物建议。在此，我们报告了一种基于Python的、与物种无关的网络工具indCAPS的开发，该工具适用于设计与indels兼容的dCAPS分析中使用的PCR引物。该工具在筛选CRISPR/Cas9诱变后的编辑事件以及在一组CRISPR介导的诱变事件中识别特定编辑事件方面应具有广泛用途。该工具在拟南芥中针对细胞分裂素受体（AHK3）的CRISPR诱变实验中进行了实地测试。该工具给出的引物成功区分了目标位点的野生型和编辑后的等位基因，并有助于分离出两个新的ahk3无效等位基因。用户可以通过http://indcaps.kieber.cloudapps.unc.edu/访问indCAPS并设计PCR引物以采用dCAPS来识别CRISPR/Cas9等位基因。
