一种用于人细胞基因组编辑效率测定和单细胞克隆筛选的 qPCR 方法。
A qPCR method for genome editing efficiency determination and single-cell clone screening in human cells.
机构信息
Shandong Provincial Key Laboratory of Animal Cell and Developmental Biology, School of Life Sciences, Shandong University, Qingdao, China.
State Key Laboratory of Microbial Technology, Shandong University, Qingdao, China.
出版信息
Sci Rep. 2019 Dec 11;9(1):18877. doi: 10.1038/s41598-019-55463-6.
CRISPR/Cas9 technology has been widely used for targeted genome modification both in vivo and in vitro. However, an effective method for evaluating genome editing efficiency and screening single-cell clones for desired modification is still lacking. Here, we developed this real time PCR method based on the sensitivity of Taq DNA polymerase to nucleotide mismatch at primer 3' end during initiating DNA replication. Applications to CRISPR gRNAs targeting EMX1, DYRK1A and HOXB13 genes in Lenti-X 293 T cells exhibited comprehensive advantages. Just in one-round qPCR analysis using genomic DNA from cells underwent CRISPR/Cas9 or BE4 treatments, the genome editing efficiency could be determined accurately and quickly, for indel, HDR as well as base editing. When applied to single-cell clone screening, the genotype of each cell colony could also be determined accurately. This method defined a rigorous and practical way in quantify genome editing events.
CRISPR/Cas9 技术已广泛应用于体内和体外的靶向基因组修饰。然而，目前仍然缺乏一种有效的方法来评估基因组编辑效率和筛选具有所需修饰的单细胞克隆。在这里，我们开发了一种基于 Taq DNA 聚合酶在引物 3' 端起始 DNA 复制时对核苷酸错配的敏感性的实时 PCR 方法。该方法应用于靶向 Lenti-X 293T 细胞中的 EMX1、DYRK1A 和 HOXB13 基因的 CRISPR gRNA，具有全面的优势。只需对经 CRISPR/Cas9 或 BE4 处理的细胞的基因组 DNA 进行一轮 qPCR 分析，就可以准确快速地确定缺失、HDR 和碱基编辑等的基因组编辑效率。当应用于单细胞克隆筛选时，还可以准确地确定每个细胞菌落的基因型。该方法为定量基因组编辑事件定义了一种严格而实用的方法。
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