Simultaneous precise editing of multiple genes in human cells
Simultaneous precise editing of multiple genes in human cells
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DOI：
10.1093/nar/gkz669
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发表时间：
2019-11-04
影响因子：
14.9
通讯作者：
Paeaebo, Svante
中科院分区：
文献类型：
--
作者：
Riesenberg, Stephan;Chintalapati, Manjusha;Paeaebo, Svante
当CRISPR将双链断裂引入基因组时，它们要么通过非同源末端连接（NHEJ）修复，这通常导致插入或缺失（indels），要么通过同源定向修复（HDR）修复，如果提供了供体寡核苷酸，则允许引入精确的核苷酸替换。由于NHEJ比HDR更有效，实现精确基因组编辑的频率非常低，因此迄今为止不可能同时编辑多个基因。在这里，我们在人类PRKDC基因中引入了一个突变，该突变消除了dna依赖性蛋白激酶催化亚基（DNA-PKcs）的激酶活性。无论细胞类型和使用的CRISPR酶如何，这都会导致HDR的增加，有时允许细胞群中87%的染色体被精确编辑。它还允许在同一细胞中同时精确编辑多达四个基因（8条染色体）。激酶抑制剂M3814对DNA-PKcs的短暂抑制同样能够增强精确的基因组编辑。
When double-strand breaks are introduced in a genome by CRISPR they are repaired either by non-homologous end joining (NHEJ), which often results in insertions or deletions (indels), or by homology-directed repair (HDR), which allows precise nucleotide substitutions to be introduced if a donor oligonucleotide is provided. Because NHEJ is more efficient than HDR, the frequency with which precise genome editing can be achieved is so low that simultaneous editing of more than one gene has hitherto not been possible. Here, we introduced a mutation in the human PRKDC gene that eliminates the kinase activity of the DNA-dependent protein kinase catalytic subunit (DNA-PKcs). This results in an increase in HDR irrespective of cell type and CRISPR enzyme used, sometimes allowing 87% of chromosomes in a population of cells to be precisely edited. It also allows for precise editing of up to four genes simultaneously (8 chromosomes) in the same cell. Transient inhibition of DNA-PKcs by the kinase inhibitor M3814 is similarly able to enhance precise genome editing.