Speed genome editing by transient CRISPR/Cas9 targeting and large DNA fragment deletion
Speed genome editing by transient CRISPR/Cas9 targeting and large DNA fragment deletion
复制标题
通过瞬时 CRISPR/Cas9 靶向和大 DNA 片段删除加速基因组编辑
DOI：
10.1016/j.jbiotec.2018.06.308
复制
发表时间：
2018-09-10
影响因子：
4.1
通讯作者：
Zhang, Lichen
中科院分区：
文献类型：
--
作者：
Luo, Jing;Lu, Liaoxun;Zhang, Lichen
CRISPR/CAS9系统极大地促进了细胞系和模式生物的基因工程。然而，在细胞系中，由于分离突变克隆的困难和复杂的基因分型，获得理想的突变克隆仍然是劳动密集型和耗时的。在这项研究中，我们验证了荧光蛋白报告程序辅助细胞分选，使突变细胞能够分离出最大的多样性。我们进一步应用两个光谱不同的荧光蛋白DsRed2和ECFP作为报告分子，进行独立的CRISPR/Cas9介导的靶向，这允许对突变细胞进行一细胞一孔的分选。由于CRISPR/Cas9系统具有双报告的超高效率，以及由于独立的位点切割而导致的大片段缺失，因此可以很容易地用常规的PCR来鉴定单克隆性突变细胞。在本文提出的快速基因组编辑方法中，不需要复杂的基因分型方法来鉴定CRISPR/Cas9基因组编辑后的功能缺失突变，并且在转染后不到一个月的时间内就可以获得理想的功能缺失突变克隆。
Genetic engineering of cell lines and model organisms has been facilitated enormously by the CRISPR/Cas9 system. However, in cell lines it remains labor intensive and time consuming to obtain desirable mutant clones due to the difficulties in isolating the mutated clones and sophisticated genotyping. In this study, we have validated fluorescent protein reporter aided cell sorting which enables the isolation of maximal diversity in mutant cells. We further applied two spectrally distinct fluorescent proteins DsRed2 and ECFP as reporters for independent CRISPR/Cas9 mediated targeting, which allows for one-cell-one-well sorting of the mutant cells. Because of ultra-high efficiency of the CRISPR/Cas9 system with dual reporters and large DNA fragment deletion resulting from independent loci cleavage, monoclonal mutant cells could be easily identified by conventional PCR. In the speed genome editing method presented here, sophisticated genotyping methods are not necessary to identify loss of function mutations after CRISPR/Cas9 genome editing, and desirable loss of function mutant clones could be obtained in less than one month following transfection.