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Modification and Application of CRIS...

Ma, Xun,

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Modification and Application of CRISPR/Cas9 Technology to Study Stem Cell Differentiation /

Record Type:	Electronic resources : Monograph/item
Title/Author:	Modification and Application of CRISPR/Cas9 Technology to Study Stem Cell Differentiation // Xun Ma.
Author:	Ma, Xun,
Description:	1 electronic resource (161 pages)
Notes:	Source: Dissertations Abstracts International, Volume: 81-10, Section: B.
Contained By:	Dissertations Abstracts International81-10B.
Subject:	Cellular biology. -
Online resource:	https://pqdd.sinica.edu.tw/twdaoapp/servlet/advanced?query=27784023
ISBN:	9781392378243

Modification and Application of CRISPR/Cas9 Technology to Study Stem Cell Differentiation /
Ma, Xun,

Modification and Application of CRISPR/Cas9 Technology to Study Stem Cell Differentiation /Xun Ma. - 1 electronic resource (161 pages)

Source: Dissertations Abstracts International, Volume: 81-10, Section: B.

The CRISPR/Cas9 technology has demonstrated great potentials in genome editing with its superior efficiency and flexibility and it has been widely applied for various studies. To explore its capacity in delineating sophisticated gene functions during cellular differentiation, we initiated this project with two parts of works.In the first part, we developed an inducible Ert2-Cas9 system to render controllable genome editing. We showed that the Ert2-Cas9 showed functional activity in the presence of tamoxifen. Based on similar design, we established the Ert2-Cpf1 system. Further, we introduced Cre/LoxP system as a secondary controller to overcome the Ert2 leakage problem and enhance the inducible feature, termed as TD-Ert2-Cas9. By this modification, the Cas9 function was controlled in both transcriptional and spatial level. We established HEPA1-6 and Tree shrew TD-Ert2-Cas9 expressing cell lines and proved the fast-responsive and efficient genome editing activity of Ert2-Cas9. These results suggest Ert2-Cas9 system is potential to be used for gene editing studies In the second part, to elaborate the functions of β-catenin during the definitive endoderm (DE) differentiation from human embryonic stem cell (hESC), we employed CRISPR/Cas9 technology to establish β-catenin knock-out hESC. By performing the DE differentiation using β-catenin knock-out cell with or without rescue, we found that, the expression of β-catenin should be maintained at proper level in order to support successful DE differentiation. Also, truncated form of β-catenin or γ-catenin, could partially rescue the differentiation. To further investigate the role of non-canonical Wnt signaling pathway during DE differentiation, WNT5A and ROR2 knock-out hESC lines were establish. Our findings provide new information for deeper understanding of the relevance between Wnt signaling pathway and hESC DE differentiation.Collectively, we established new tools based on CRISPR/Cas9 technology and showed that they are valuable to study sophisticated gene functions, especially during dynamic cellular differentiation. This work provides a solid foundation to obtain further insights into the molecular regulation underlying stem cell fate control in future.

English

ISBN: 9781392378243Subjects--Topical Terms:

3172791
Cellular biology.
Subjects--Index Terms:

Genome editing

Modification and Application of CRISPR/Cas9 Technology to Study Stem Cell Differentiation /
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520 $a The CRISPR/Cas9 technology has demonstrated great potentials in genome editing with its superior efficiency and flexibility and it has been widely applied for various studies. To explore its capacity in delineating sophisticated gene functions during cellular differentiation, we initiated this project with two parts of works.In the first part, we developed an inducible Ert2-Cas9 system to render controllable genome editing. We showed that the Ert2-Cas9 showed functional activity in the presence of tamoxifen. Based on similar design, we established the Ert2-Cpf1 system. Further, we introduced Cre/LoxP system as a secondary controller to overcome the Ert2 leakage problem and enhance the inducible feature, termed as TD-Ert2-Cas9. By this modification, the Cas9 function was controlled in both transcriptional and spatial level. We established HEPA1-6 and Tree shrew TD-Ert2-Cas9 expressing cell lines and proved the fast-responsive and efficient genome editing activity of Ert2-Cas9. These results suggest Ert2-Cas9 system is potential to be used for gene editing studies In the second part, to elaborate the functions of β-catenin during the definitive endoderm (DE) differentiation from human embryonic stem cell (hESC), we employed CRISPR/Cas9 technology to establish β-catenin knock-out hESC. By performing the DE differentiation using β-catenin knock-out cell with or without rescue, we found that, the expression of β-catenin should be maintained at proper level in order to support successful DE differentiation. Also, truncated form of β-catenin or γ-catenin, could partially rescue the differentiation. To further investigate the role of non-canonical Wnt signaling pathway during DE differentiation, WNT5A and ROR2 knock-out hESC lines were establish. Our findings provide new information for deeper understanding of the relevance between Wnt signaling pathway and hESC DE differentiation.Collectively, we established new tools based on CRISPR/Cas9 technology and showed that they are valuable to study sophisticated gene functions, especially during dynamic cellular differentiation. This work provides a solid foundation to obtain further insights into the molecular regulation underlying stem cell fate control in future.
520 $a CRISPR 基因編輯系統起源於古細菌內的病毒防禦系統。該系統利用短片段RNA 序列識別特定的基因序列，並介導獨特的核酸酶對特定序列進行切割，從而激活 DNA 修復系統進行修復。來自S. pyogenes 的Cas9 核酸酶被首先發現並應用於基因編輯研究。這壹工具平臺的建立大大加快了相關領域的，特別是幹細胞領域的研究進展。在第壹部分的研究中，我們在CRISPR/Cas9 系統的基礎上，引入了tamoxifen 調控系統和Cre/LoxP 重組酶技術, 來獲得高效可控的基因編輯工具Ert2-Cas9 和改進的TD-Ert2-Cas9。首先我們在不同細胞系（HEK293T，LO2 細胞系）中證實了 Ert2-Cas9 的有效性和可控性。接著，我們利用CRISPR/Cas9 系統將改進的TDErt2- Cas9 敲入不同細胞系（小鼠HEPA1-6 細胞系，樹鼩原代上皮細胞系）的基因組內，獲得可以穩定表達這壹核酸酶體系的新細胞系。並通過tamoxifen 和Cre 重組酶進壹步證實了細胞系的有效性和高度可控的特性。利用這壹工具，我們可以實現短時間（＜2 小時）的高效基因編輯，大大提高了時效性，為進壹步的研究提供了技術支持。第二部分研究中，我們利用CRISPR/Cas9 系統在人胚胎幹細胞中敲除了基因β- catenin，通過體外細胞分化模型來研究該基因在限定性內胚層分化過程中的作用。限定性內胚層是胚胎發育到內胚層的關鍵階段，研究表明Wnt 信號通路在限定性內胚層的形成過程中起到關鍵作用，經典Wnt 信號通路的關鍵基因β-catenin 的表達水平會影響細胞向內胚層的分化能力。利用這壹基因敲除細胞系，我們利用腺病毒載體表達不同功能節段性的β-catenin，發現成功的限定性內胚層分化需要合適水平的β-catenin 表達。但是，功能節段性的β-catenin 表達依然可以激活中等至幾乎完全的限定性內胚層分化。具有類似β-catenin 功能的γ-catenin 也可以引起類似的細胞分化表型。進壹步的，基於我們既往的對非經典Wnt 信號通路的研究結果，我們用同樣的方法對其關鍵基因進行了敲除，來進壹步明確這壹信號通路對限定性內胚層分化的影響。總的來講，CRISPR/Cas9 系統為我們提供了研究基因功能的強大工具平臺，並且本身具有很強的可操作性。.
546 $a English
590 $a School code: 1307
650 4 $a Cellular biology. $3 3172791
650 4 $a Genetics. $3 530508
653 $a Genome editing
653 $a Cas9
653 $a β-catenin
690 $a 0379
690 $a 0369
710 2 $a The Chinese University of Hong Kong (Hong Kong). $e degree granting institution. $3 3765695
720 1 $a Feng, Bo $e degree supervisor.
773 0 $t Dissertations Abstracts International $g 81-10B.
790 $a 1307
791 $a Ph.D.
792 $a 2019
856 4 0 $u https://pqdd.sinica.edu.tw/twdaoapp/servlet/advanced?query=27784023