Friedman Clayton E, Fayer Shawn, Pendyala Sriram, Chien Wei-Ming, Tran Linda, Chao Leslie, Mckinstry Ashley, Karbassi Elaheh, Fenix Aidan M, Loiben Alexander, Murry Charles E, Starita Lea M, Fowler Douglas M, Yang Kai-Chun
bioRxiv. 2023 Jan 26:2023.01.25.525582. doi: 10.1101/2023.01.25.525582.
Standard transgenic cell line generation requires screening 100-1000s of colonies to isolate correctly edited cells. We describe CR ISPR a On- T arget E diting R etrieval (CRaTER) which enriches for cells with on-target knock-in of a cDNA-fluorescent reporter transgene by transient activation of the targeted locus followed by flow sorting to recover edited cells. We show CRaTER recovers rare cells with heterozygous, biallelic-editing of the transcriptionally-inactive locus in human induced pluripotent stem cells (hiPSCs), enriching on average 25-fold compared to standard antibiotic selection. We leveraged CRaTER to enrich for heterozygous knock-in of a library of single nucleotide variants (SNVs) in , a gene in which missense mutations cause cardiomyopathies, and recovered hiPSCs with 113 different SNVs. We differentiated these hiPSCs to cardiomyocytes and show MYH7 fusion proteins can localize as expected. Thus, CRaTER substantially reduces screening required for isolation of gene-edited cells, enabling generation of transgenic cell lines at unprecedented scale.
标准转基因细胞系的生成需要筛选100到1000个菌落以分离出正确编辑的细胞。我们描述了CRISPR靶向编辑检索（CRaTER）方法，该方法通过瞬时激活目标位点，然后进行流式分选以回收编辑后的细胞，从而富集具有cDNA荧光报告转基因靶向敲入的细胞。我们展示了CRaTER能够在人类诱导多能干细胞（hiPSC）中回收具有转录失活位点杂合、双等位基因编辑的稀有细胞，与标准抗生素筛选相比，平均富集了25倍。我们利用CRaTER富集了一个单核苷酸变体（SNV）文库的杂合敲入，该文库位于一个基因中，该基因的错义突变会导致心肌病，并回收了具有113种不同SNV的hiPSC。我们将这些hiPSC分化为心肌细胞，并表明MYH7融合蛋白能够按预期定位。因此，CRaTER大大减少了分离基因编辑细胞所需的筛选工作，能够以前所未有的规模生成转基因细胞系。
