Zaboikin Michail, Zaboikina Tatiana, Freter Carl, Srinivasakumar Narasimhachar
Division of Hematology/Oncology, Department of Internal Medicine, Saint Louis University, Saint Louis, Missouri, United States of America.
PLoS One. 2017 Jan 17;12(1):e0169931. doi: 10.1371/journal.pone.0169931. eCollection 2017.
Genome editing using transcription-activator like effector nucleases or RNA guided nucleases allows one to precisely engineer desired changes within a given target sequence. The genome editing reagents introduce double stranded breaks (DSBs) at the target site which can then undergo DNA repair by non-homologous end joining (NHEJ) or homology directed recombination (HDR) when a template DNA molecule is available. NHEJ repair results in indel mutations at the target site. As PCR amplified products from mutant target regions are likely to exhibit different melting profiles than PCR products amplified from wild type target region, we designed a high resolution melting analysis (HRMA) for rapid identification of efficient genome editing reagents. We also designed TaqMan assays using probes situated across the cut site to discriminate wild type from mutant sequences present after genome editing. The experiments revealed that the sensitivity of the assays to detect NHEJ-mediated DNA repair could be enhanced by selection of transfected cells to reduce the contribution of unmodified genomic DNA from untransfected cells to the DNA melting profile. The presence of donor template DNA lacking the target sequence at the time of genome editing further enhanced the sensitivity of the assays for detection of mutant DNA molecules by excluding the wild-type sequences modified by HDR. A second TaqMan probe that bound to an adjacent site, outside of the primary target cut site, was used to directly determine the contribution of HDR to DNA repair in the presence of the donor template sequence. The TaqMan qPCR assay, designed to measure the contribution of NHEJ and HDR in DNA repair, corroborated the results from HRMA. The data indicated that genome editing reagents can produce DSBs at high efficiency in HEK293T cells but a significant proportion of these are likely masked by reversion to wild type as a result of HDR. Supplying a donor plasmid to provide a template for HDR (that eliminates a PCR amplifiable target) revealed these cryptic DSBs and facilitated the determination of the true efficacy of genome editing reagents. The results indicated that in HEK293T cells, approximately 40% of the DSBs introduced by genome editing, were available for participation in HDR.
使用转录激活因子样效应物核酸酶或RNA引导核酸酶进行基因组编辑，可使人们在给定的靶序列内精确构建所需的改变。基因组编辑试剂在靶位点引入双链断裂（DSB），当有模板DNA分子时，这些双链断裂随后可通过非同源末端连接（NHEJ）或同源定向重组（HDR）进行DNA修复。NHEJ修复导致靶位点出现插入缺失突变。由于来自突变靶区域的PCR扩增产物可能呈现出与从野生型靶区域扩增的PCR产物不同的熔解曲线，我们设计了一种高分辨率熔解分析（HRMA）方法，用于快速鉴定高效的基因组编辑试剂。我们还设计了TaqMan检测法，使用位于切割位点两侧的探针来区分基因组编辑后出现的野生型和突变型序列。实验表明，通过选择转染细胞以减少未转染细胞中未修饰基因组DNA对DNA熔解曲线的影响，可提高检测NHEJ介导的DNA修复的检测方法的灵敏度。在基因组编辑时存在缺乏靶序列的供体模板DNA，通过排除由HDR修饰的野生型序列，进一步提高了检测突变DNA分子的检测方法的灵敏度。第二个与主要靶切割位点之外的相邻位点结合的TaqMan探针，用于在存在供体模板序列的情况下直接确定HDR对DNA修复的贡献。旨在测量NHEJ和HDR在DNA修复中贡献的TaqMan qPCR检测法，证实了HRMA的结果。数据表明，基因组编辑试剂可在HEK293T细胞中高效产生DSB，但其中很大一部分可能因HDR而恢复为野生型，从而被掩盖。提供供体质粒以提供HDR模板（消除可PCR扩增的靶标），揭示了这些隐蔽的DSB，并有助于确定基因组编辑试剂的真正功效。结果表明，在HEK293T细胞中，基因组编辑引入的DSB中约40%可用于参与HDR。
