Genome-wide tracking of dCas9-methyltransferase footprints.
Genome-wide tracking of dCas9-methyltransferase footprints.
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DOI：
10.1038/s41467-017-02708-5
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发表时间：
2018-02-09
影响因子：
16.6
通讯作者：
Meissner A
中科院分区：
文献类型：
--
作者：
Galonska C;Charlton J;Mattei AL;Donaghey J;Clement K;Gu H;Mohammad AW;Stamenova EK;Cacchiarelli D;Klages S;Timmermann B;Cantz T;Schöler HR;Gnirke A;Ziller MJ;Meissner A
在正常的哺乳动物发育中，胞嘧啶甲基化是必需的，并且针对基因组的特定区域。尽管通过绘制其全基因组分布取得了显着进展，但由于缺乏精确操作的工具，研究DNA甲基化对基因和基因组调控的直接贡献受到限制。因此，将CRISPR-Cas9系统的靶向能力与表观遗传修饰剂相结合引起了科学界的兴趣。与分析具有核酸酶能力的Cas9的全基因组切割相反，在高度甲基化的基因组中追踪死亡Cas9（dCas 9）甲基转移酶融合蛋白的全局活性具有挑战性。在这里，我们报告了工程化的、甲基化耗尽的但维持能力强的小鼠ES细胞系的产生和使用，并且令人惊讶地发现了dCas 9-甲基转移酶的普遍存在的核活性。随后在人体细胞中的实验完善了这些观察结果，并指出了遗传和表观遗传编辑工具之间的重要差异，需要独特的实验考虑。与甲基转移酶融合的无催化活性的Cas9已经成为一种有前途的表观基因组修饰工具。在这里，作者产生了甲基化缺失但维持能力的小鼠ES细胞系，并发现了普遍存在的脱靶活性。
In normal mammalian development cytosine methylation is essential and is directed to specific regions of the genome. Despite notable advances through mapping its genome-wide distribution, studying the direct contribution of DNA methylation to gene and genome regulation has been limited by the lack of tools for its precise manipulation. Thus, combining the targeting capability of the CRISPR–Cas9 system with an epigenetic modifier has attracted interest in the scientific community. In contrast to profiling the genome-wide cleavage of a nuclease competent Cas9, tracing the global activity of a dead Cas9 (dCas9) methyltransferase fusion protein is challenging within a highly methylated genome. Here, we report the generation and use of an engineered, methylation depleted but maintenance competent mouse ES cell line and find surprisingly ubiquitous nuclear activity of dCas9-methyltransferases. Subsequent experiments in human somatic cells refine these observations and point to an important difference between genetic and epigenetic editing tools that require unique experimental considerations. Catalytically inactive Cas9 fused to a methyltransferase has emerged as a promising epigenome modifying tool. Here the authors generate a methylation depleted but maintenance competent mouse ES cell line and find ubiquitous off-target activity.
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影响因子：
64.5
作者：
Gifford CA;Ziller MJ;Gu H;Trapnell C;Donaghey J;Tsankov A;Shalek AK;Kelley DR;Shishkin AA;Issner R;Zhang X;Coyne M;Fostel JL;Holmes L;Meldrim J;Guttman M;Epstein C;Park H;Kohlbacher O;Rinn J;Gnirke A;Lander ES;Bernstein BE;Meissner A
通讯作者：
Meissner A
影响因子：
64.5
作者：
Hsu PD;Lander ES;Zhang F
通讯作者：
Zhang F
影响因子：
64.5
作者：
Okano, M;Bell, DW;Li, E
通讯作者：
Li, E
影响因子：
12.3
作者：
Sun D;Xi Y;Rodriguez B;Park HJ;Tong P;Meong M;Goodell MA;Li W
通讯作者：
Li W
影响因子：
14.9
作者：
Stepper P;Kungulovski G;Jurkowska RZ;Chandra T;Krueger F;Reinhardt R;Reik W;Jeltsch A;Jurkowski TP
通讯作者：
Jurkowski TP