Generation and validation of versatile inducible CRISPRi embryonic stem cell and mouse model.
Generation and validation of versatile inducible CRISPRi embryonic stem cell and mouse model.
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多功能诱导型 CRISPRi 胚胎干细胞和小鼠模型的生成和验证
DOI：
10.1371/journal.pbio.3000749
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发表时间：
2020-11
期刊：
影响因子：
9.8
通讯作者：
Wu X
中科院分区：
文献类型：
--
作者：
Li R;Xia X;Wang X;Sun X;Dai Z;Huo D;Zheng H;Xiong H;He A;Wu X
簇状规则间隔短回文重复(CRISPR)-CRISPR相关的(Cas)9已经被广泛应用，远远超出了基因组编辑的范围。失活的Cas9(DCas9)与转录效应器的融合使对表观基因组的询问和基因表达的控制成为可能。然而，dCas9-融合基因的大小阻碍了其在体细胞组织中的应用。在这里，我们通过在小鼠胚胎干细胞(IKRAb ESC)中转基因表达多西环素(Dox)诱导的dCas9-KRAb来建立一个健壮的CRISPR干扰(CRISPRi)系统。在引入特定的单引导RNAs(SgRNAs)后，诱导的dCas9-KRAb有效地维持了基因的失活，尽管它适度下调了活性基因的表达。在细胞分化或重编程期间适当的Dox添加时间允许我们研究或筛选时空激活的启动子或增强子，从而研究或筛选基因功能。此外，将胚胎干细胞用于胚泡注射，我们建立了一个iKRAB敲入(KI)小鼠模型，该模型能够通过简单的gRNAs转导来关闭基因表达和功能丧失(LOF)的体外和体内研究。因此，我们的可诱导CRISPRi ESC系和KI小鼠为发育或疾病背景下的功能询问和高通量筛选特定基因和潜在调控元件提供了通用和方便的平台。这项研究描述了多西环素可诱导的CRISPRi胚胎干细胞系和敲入小鼠模型的建立，它们共同为功能询问和高通量筛选参与发育或疾病的特定基因和潜在调控元件提供了通用和方便的平台。
Clustered regularly interspaced short palindromic repeat (CRISPR)-CRISPR-associated (Cas) 9 has been widely used far beyond genome editing. Fusions of deactivated Cas9 (dCas9) to transcription effectors enable interrogation of the epigenome and controlling of gene expression. However, the large transgene size of dCas9-fusion hinders its applications especially in somatic tissues. Here, we develop a robust CRISPR interference (CRISPRi) system by transgenic expression of doxycycline (Dox) inducible dCas9-KRAB in mouse embryonic stem cells (iKRAB ESC). After introduction of specific single-guide RNAs (sgRNAs), the induced dCas9-KRAB efficiently maintains gene inactivation, although it modestly down-regulates the expression of active genes. The proper timing of Dox addition during cell differentiation or reprogramming allows us to study or screen spatiotemporally activated promoters or enhancers and thereby the gene functions. Furthermore, taking the ESC for blastocyst injection, we generate an iKRAB knock-in (KI) mouse model that enables the shutdown of gene expression and loss-of-function (LOF) studies ex vivo and in vivo by a simple transduction of gRNAs. Thus, our inducible CRISPRi ESC line and KI mouse provide versatile and convenient platforms for functional interrogation and high-throughput screens of specific genes and potential regulatory elements in the setting of development or diseases. This study describes the generation of a doxycycline-inducible CRISPRi embryonic stem cell line and knock-in mouse model that together provide versatile and convenient platforms for functional interrogation and high-throughput screens of specific genes and potential regulatory elements involved in development or disease.
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DOI：
10.1126/science.aah7111
发表时间：
2017-01-06
期刊：
Science (New York, N.Y.)
影响因子：
--
作者：
Liu SJ;Horlbeck MA;Cho SW;Birk HS;Malatesta M;He D;Attenello FJ;Villalta JE;Cho MY;Chen Y;Mandegar MA;Olvera MP;Gilbert LA;Conklin BR;Chang HY;Weissman JS;Lim DA
通讯作者：
Lim DA
影响因子：
64.5
作者：
Phillips-Cremins JE;Sauria ME;Sanyal A;Gerasimova TI;Lajoie BR;Bell JS;Ong CT;Hookway TA;Guo C;Sun Y;Bland MJ;Wagstaff W;Dalton S;McDevitt TC;Sen R;Dekker J;Taylor J;Corces VG
通讯作者：
Corces VG
影响因子：
64.5
作者：
Hsu PD;Lander ES;Zhang F
通讯作者：
Zhang F
影响因子：
23.9
作者：
Nichols, Jennifer;Smith, Austin
通讯作者：
Smith, Austin
影响因子：
64.5
作者：
Gasperini, Molly;Hill, Andrew J.;Shendure, Jay
通讯作者：
Shendure, Jay