Nussbaum Lea, Telenius Jelena M, Hill Stephanie, Hirschfeld Priscila P, Suciu Maria C, Downes Damien J, Hughes Jim R
MRC Molecular Haematology Unit, Weatherall Institute of Molecular Medicine, Oxford OX3 9DU, UK.
MRC WIMM Center for Computational Biology, Weatherall Institute of Molecular Medicine, Oxford OX3 9DU, UK.
Methods Protoc. 2018 Aug 1;1(3):29. doi: 10.3390/mps1030029.
The emergence in recent years of DNA editing technologies-Zinc finger nucleases (ZFNs), transcription activator-like effector (TALE) guided nucleases (TALENs), clustered regularly interspaced short palindromic repeats (CRISPR)/Cas family enzymes, and Base-Editors-have greatly increased our ability to generate hundreds of edited cells carrying an array of alleles, including single-nucleotide substitutions. However, the infrequency of homology-dependent repair (HDR) in generating these substitutions in general requires the screening of large numbers of edited cells to isolate the sequence change of interest. Here we present a high-throughput method for the amplification and barcoding of edited loci in a 96-well plate format. After barcoding, plates are indexed as pools which permits multiplexed sequencing of hundreds of clones simultaneously. This protocol works at high success rate with more than 94% of clones successfully genotyped following analysis.
近年来，DNA编辑技术——锌指核酸酶（ZFNs）、转录激活样效应物（TALE）引导的核酸酶（TALENs）、成簇规律间隔短回文重复序列（CRISPR）/Cas家族酶以及碱基编辑器的出现，极大地提高了我们生成数百个携带一系列等位基因（包括单核苷酸替换）的编辑细胞的能力。然而，一般情况下，在产生这些替换时同源依赖性修复（HDR）的频率较低，这就需要筛选大量的编辑细胞以分离出感兴趣的序列变化。在此，我们展示了一种以96孔板形式对编辑位点进行扩增和条形码标记的高通量方法。条形码标记后，各板作为混合样本进行索引，从而允许同时对数百个克隆进行多重测序。按照此方案操作成功率很高，分析后超过94%的克隆能够成功进行基因分型。
