CRISPR-Cas9 nuclear dynamics and target recognition in living cells.
CRISPR-Cas9 nuclear dynamics and target recognition in living cells.
复制标题
DOI：
10.1083/jcb.201604115
复制
发表时间：
2016-08-29
期刊：
影响因子：
--
通讯作者：
Pederson T
中科院分区：
文献类型：
--
作者：
Ma H;Tu LC;Naseri A;Huisman M;Zhang S;Grunwald D;Pederson T
CRISPR Cas9-guide RNA复合物如何导航细胞核并询问基因组还不清楚。Ma等人在活细胞中追踪这些复合物，发现引导种子区域中的突变显著降低了复合物的靶停留时间，同时相应地损害了切割。细菌CRISPR-Cas9系统已被重新用于真核细胞中的基因组工程、转录调节和染色体成像。然而，成簇的规则间隔短回文重复序列（CRISPR）相关蛋白9（Cas9）指导RNA和靶询问的核动力学在活细胞中没有很好地定义。在这里，我们部署了一个双色CRISPR系统来直接测量Cas9和指导RNA的稳定性。我们发现Cas9对于向导RNA稳定性是必不可少的，并且核Cas9-向导RNA复合物水平限制了靶向效率。光漂白测量后的荧光恢复显示，指导RNA种子序列中的单个错配以核苷酸同一性和位置依赖性方式将靶停留时间从>3小时降低至低至<2分钟。我们进一步表明，目标驻留的持续时间与切割活性相关。这些结果表明，CRISPR通过彻底改变停留时间来区分真正的基因组编辑靶点与不匹配的靶点。
How CRISPR Cas9–guide RNA complexes navigate the nucleus and interrogate the genome is not well understood. Ma et al. track these complexes in live cells and find that mutations in the guide seed region significantly reduced the complex’s target residence time, with a commensurate impairment of cleavage. The bacterial CRISPR-Cas9 system has been repurposed for genome engineering, transcription modulation, and chromosome imaging in eukaryotic cells. However, the nuclear dynamics of clustered regularly interspaced short palindromic repeats (CRISPR)–associated protein 9 (Cas9) guide RNAs and target interrogation are not well defined in living cells. Here, we deployed a dual-color CRISPR system to directly measure the stability of both Cas9 and guide RNA. We found that Cas9 is essential for guide RNA stability and that the nuclear Cas9–guide RNA complex levels limit the targeting efficiency. Fluorescence recovery after photobleaching measurements revealed that single mismatches in the guide RNA seed sequence reduce the target residence time from >3 h to as low as <2 min in a nucleotide identity- and position-dependent manner. We further show that the duration of target residence correlates with cleavage activity. These results reveal that CRISPR discriminates between genuine versus mismatched targets for genome editing via radical alterations in residence time.
登录
查看更多内容
CRIS/CAS9活性的生物物理模型，用于基因组编辑和基因调节的合理设计。
影响因子：
4.3
作者：
Farasat I;Salis HM
通讯作者：
Salis HM
影响因子：
8.8
作者：
Rutkauskas, Marius;Sinkunas, Tomas;Seidel, Ralf
通讯作者：
Seidel, Ralf
影响因子：
46.9
作者：
Doench JG;Fusi N;Sullender M;Hegde M;Vaimberg EW;Donovan KF;Smith I;Tothova Z;Wilen C;Orchard R;Virgin HW;Listgarten J;Root DE
通讯作者：
Root DE
影响因子：
56.9
作者：
Jiang, Fuguo;Zhou, Kaihong;Doudna, Jennifer A.
通讯作者：
Doudna, Jennifer A.
影响因子：
56.9
作者：
Knight, Spencer C.;Xie, Liangqi;Tjian, Robert
通讯作者：
Tjian, Robert