遗传 ›› 2020, Vol. 42 ›› Issue (1): 18-31.doi: 10.16288/j.yczz.19-246
收稿日期:
2019-08-22
修回日期:
2019-11-18
出版日期:
2020-01-20
发布日期:
2019-11-19
通讯作者:
吴强
E-mail:qwu123@gmail.com
作者简介:
刘沛峰,博士研究生,专业方向:生物学。E-mail: lpfmail@foxmail.com
基金资助:
Received:
2019-08-22
Revised:
2019-11-18
Online:
2020-01-20
Published:
2019-11-19
Contact:
Wu Qiang
E-mail:qwu123@gmail.com
Supported by:
摘要:
CRISPR/Cas9系统在基因编辑方面具有巨大优势,能够低成本、可编程、方便快捷地用于动物、植物以及微生物的基因组靶向编辑和功能改造。三维基因组学是近年来兴起的一门研究染色质高级结构动态调控及基因组生物学功能的交叉学科。在三维基因组研究中,通常采用对DNA片段进行基因编辑以模拟基因组结构性变异,标记特定DNA片段,进而研究调控元件对于基因调控、细胞分化、组织发生、器官形成、个体发育的影响,最终阐明三维基因组的组装调控机制和生物学功能。因此,CRISPR及其衍生技术为研究三维基因组提供了极好的遗传学工具。本文主要综述了CRISPR片段编辑及其衍生技术在三维基因组调控与功能研究中的应用,以期为后续研究工作提供理论参考以及新的研究思路。
刘沛峰, 吴强. CRISPR/Cas9基因编辑在三维基因组研究中的应用[J]. 遗传, 2020, 42(1): 18-31.
Peifeng Liu, Qiang Wu. Probing 3D genome by CRISPR/Cas9[J]. Hereditas(Beijing), 2020, 42(1): 18-31.
图1
基因组编辑技术 A:基因打靶技术原理。基因打靶技术利用同源重组的方法将目的片段导入基因组。B:锌指核酸酶基因组编辑技术原理。将特异性识别DNA序列的锌指结构串联,与Fok Ⅰ核酸内切酶融合,实现基因组靶向切割。再利用DNA修复系统实现基因组编辑。C:类转录激活因子效应物核酸酶基因组编辑技术原理。将特异性识别DNA序列的类转录激活因子效应物串联,与Fok Ⅰ核酸内切酶融合,实现基因组靶向切割,再利用DNA修复系统实现基因组编辑。D:CRISPR/Cas9基因组编辑技术原理。Cas9蛋白可以被sgRNA引导至基因组特定位点,Cas9不具有核酸外切酶活性,仅仅具有核酸内切酶活性。Cas9的HNH和RuvC结构域分别催化互补链和非互补链的核酸内切反应造成DNA的双链断裂。HNH结构域造成的断裂位点位于PAM序列上游第3与第4个核苷酸之间,而RuvC结构域在PAM序列上游可造成多达4种不同的DNA断裂。"
图2
CRISPR在三维基因组研究中的技术方法 A:CRISPR介导的DNA片段编辑。通过一对sgRNA介导Cas9在基因组中进行靶向双位点切割,可以造成基因组片段删除、反转、重复或成环等现象,为三维基因组研究提供模型。B:CRISPR介导的基因组位点可视化。通过在sgRNA上加入MS2与PP7等茎环结构,招募融合了荧光蛋白的MCP、PCP等蛋白,将荧光信号特异性地标记在特定的基因组位点。C:CRISPR介导的基因组位点重定位。利用植物激素ABA介导的PYL1与ABI1的相互作用,将PYL1和ABI1分别结合在不同系统的dCas9或其他核体特有的蛋白上,可以将特定的基因组位点定位到新的目标位点。D:以CRISPR为平台的靶向效应系统。将激活或抑制转录以及表观遗传修饰相关蛋白结合在dCas9上,可以特异性地使基因组特定位点的转录水平以及表观遗传学修饰改变。"
图3
人类原钙粘蛋白基因簇结构以及CRISPR介导的基因组编辑 A:人类原钙粘蛋白基因簇。人类原钙粘蛋白基因座包含α、β、γ 3个基因簇,并且在基因启动子与增强子区域包含大量的CTCF位点(CTCF-binding site, CBS)。通过方向性结合这些CBSs,CTCF可以介导染色质环形成,调控原钙粘蛋白基因表达。B:CRISPR系统介导的基因组元件反转改变染色质拓扑结构。基因组折叠的染色质环挤出模型(chromatin loop extrusion):沿染色质滑动的cohesin可以将染色质挤出形成染色质环,而cohesin的滑动会被一对方向收敛或者说正向-反向(convergent or forward-reverse)的CBS上的CTCF阻滞,这使得染色质环挤出的进程终止,两个CBS在三维空间上被拉近。利用CRISPR/Cas9系统反转CBS方向能够改变染色质环化方向,证明了CTCF位点的方向性在三维基因组折叠中的关键作用。"
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