Improved Genome Editing through Inhibition of FANCM and Members of the BTR Dissolvase Complex
Improved Genome Editing through Inhibition of FANCM and Members of the BTR Dissolvase Complex
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DOI：
10.1016/j.ymthe.2020.10.020
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发表时间：
2021-03-03
影响因子：
12.4
通讯作者：
Kay, Mark A.
中科院分区：
文献类型：
--
作者：
Alencastro, Gustavo de;Puzzo, Francesco;Kay, Mark A.
重组腺相关病毒（rAAV）载体具有能够进行基因组靶向整合（TI）而不诱导双链断裂（DSB）的独特性质。为了提高我们对AAV介导的TI背后的机制的理解并提高其效率，我们使用无启动子的AAV同源重组（AAVHR）载体系统在人类细胞中进行了无偏倚的遗传筛选。我们鉴定了在不同培养的人细胞中，范可尼贫血互补组M（FANCM）蛋白的抑制将AAV-HR介导的TI效率提高了6至9倍。FANCM和也与FANCM复合物相关的两种蛋白质，RecQ介导的基因组不稳定性1（RMI 1）和来自BLM-拓扑异构酶IIIc（TOP 3A）-RMI（BTR）溶解酶复合物的Bloom DNA解旋酶（BLM）（RMI 1，也已在我们的筛选中鉴定）的组合敲低导致AAV-HR介导的TI增强高达相似的17倍。在核酸酶（CRISPR-Cas9）存在下，AAV-HR介导的TI在FANCM和RMI 1敲除细胞中也分别增加了1.5至2倍。此外，在人CD 34（+）造血干细胞和祖细胞（HSPC）中敲低FANCM使AAV-HR介导的TI增加了约3.5倍。这项研究扩展了我们对AAV介导的TI相关机制的了解，并强调了可能用于未来改善AAV-HR介导的TI的新途径。
Recombinant adeno-associated virus (rAAV) vectors have the unique property of being able to perform genomic targeted integration (TI) without inducing a double-strand break (DSB). In order to improve our understanding of the mechanism behind TI mediated by AAV and improve its efficiency, we performed an unbiased genetic screen in human cells using a promoterless AAV-homologous recombination (AAVHR) vector system. We identified that the inhibition of the Fanconi anemia complementation group M (FANCM) protein enhanced AAV-HR-mediated TI efficiencies in different cultured human cells by similar to 6- to 9-fold. The combined knockdown of the FANCM and two proteins also associated with the FANCM complex, RecQ-mediated genome instability 1 (RMI1) and Bloom DNA helicase (BLM) from the BLM-topoisomerase IIIc (TOP3A)-RMI (BTR) dissolvase complex (RMI1, having also been identified in our screen), led to the enhancement of AAV-HR-mediated TI up to similar to 17 times. AAV-HR-mediated TI in the presence of a nuclease (CRISPR-Cas9) was also increased by similar to 1.5- to 2-fold in FANCM and RMI1 knockout cells, respectively. Furthermore, knockdown of FANCM in human CD34(+) hematopoietic stem and progenitor cells (HSPCs) increased AAV-HR-mediated TI by similar to 3.5-fold. This study expands our knowledge on the mechanisms related to AAV-mediated TI, and it highlights new pathways that might be manipulated for future improvements in AAV-HR-mediated TI.