CRISPR/Cas9系統創建之小鼠體細胞突變在癌症研究的應用

Abstract

癌症為一個異質性高的多基因疾病，目前癌症基因體相關研究已經找到為數眾多的癌症基因突變，而許多突變都尚未在活體內驗證它們的生物活性。在近期興起的CRISPR/Cas9系統讓我們擁有了前所未有的高效率精確修改基因體的方法，該技術的發展大幅地加速了體細胞基因編修的實現，讓我們能夠在活體內針對特定標的癌症基因製造突變細胞群，在不必經由過多冗長、費力的動物交配、繁殖之下，可以在原位突變的體細胞群上進行遺傳篩選或檢驗不同癌症基因突變事件間對於癌症起始、進程的合作關係。在這個研究中，我提出了一個結合誘導性Cas9效應物以及CRISPR誘變物 (ICE CRIM) 的轉殖基因小鼠模式，在這個小鼠模式中，CRISPR/Cas9系統會因應環化重組酶 (Cre) 的表現而活化，進而誘導活體內體細胞突變事件的發生。我選擇了著名的抑癌基因Trp53以及兩個重要的DNA錯配修復基因 (Mlh1跟Msh2) 做為此研究中進行體細胞突變的目標基因。我已成功建造並繁殖三個品系的Trp53複合轉基因小鼠，分別針對Trp53轉譯起始密碼以及核酸辨識結合區進行破壞，以及一個同時表現針對Mlh1以及Msh2單一嚮導核醣核酸 (sgRNA) 的LS轉基因小鼠品系。Surveyor分析跟擴增子定序技術在此被用來驗證活體內各個目標基因的編修效率。同時，擴增子定序技術也用來定義所產生的突變等位基因系列。藉由搭配多種不同品系的環化重組酶小鼠 (Sox2-Cre, Alb-Cre, 以及Zp3-Cre小鼠)，所建立的ICE CRIM轉殖基因能受到活化，並且在體細胞或者生殖細胞產生目標基因的突變等位基因。此研究提供了直接的實驗證據證明活化的ICE CRIM轉殖基因可以在一個細胞中同時針對目標基因的兩個等位基因進行突變。當所建立的CRISPR誘變物帶有多個嚮導核醣核酸時，活化的ICE CRIM轉殖基因也可以達成同時破壞多個目標基因的目標。此研究的小鼠模式能夠在活體內建立目標基因的突變庫，以供後續在原位進行各個突變的功能性篩選。在所建立的小鼠模式中，CRISPR/Cas9系統藉由誘導的不精確插入缺失事件，產生了多種不同的突變等位基因，而這些序列上的標記提供了一個在活體內做純系分析的工具，藉此特徵，我的實驗成果也提供了支持單一細胞來源的血液腫瘤生成的證據。另外，此研究的實驗成果也驗證了在Trp53突變的遺傳背景下，DNA錯配修復的缺失會加速腫瘤生成的現象。CRISPR/Cas9系統的應用讓我們在未來能夠以基因體序列為基礎，有系統地針對多個癌症相關基因去設計癌症研究，進而了解不同癌症相關基因對於癌症起始、進程的合作關係。

Cancer is a multistep, heterogeneous, genetic disease. Numerous newly identified cancer gene mutations (and the interactions among them) derived from cancer genome projects have yet been functionally illustrated. The CRISPR/Cas9 technology allows somatic genome editing to create mutant pools of target gene(s). When selective pressure is applied, an in situ somatic genetic screen can be performed. Cooperative genetic interactions may also be revealed at the cellular level without extensive breeding between different germline mutant animals. In this study, I proposed a transgenic inducible Cas9 effector/CRISPR mutagen (ICE CRIM) mouse model in which CRISPR/Cas9-mediated somatic mutagenesis events can occur in response to Cre expression. I chose the well-known tumor suppressor gene, Trp53, and two important DNA mismatch repair genes, Mlh1 and Msh2, to be our somatic mutagenesis targets. Three Trp53 ICE CRIM transgenic mouse lines, targeting exon 2 or exon 7 of Trp53, as well as a LS CRIM transgenic mouse line, expressing sgRNAs targeting Mlh1 and Msh2, were generated. Surveyor assay and amplicon-based sequencing were performed to validate the editing efficiency of each target gene. Additionally, amplicon-based sequencing was also used to identify the mutant allelic series. By crossing with different Cre lines (Sox2-Cre, Alb-Cre, and Zp3-Cre), the ICE CRIM allele was activated to generate somatic- and/or germline-mutant alleles of target gene(s). I provided experimental evidence to show that both targeted alleles within a cell can be mutated by an activated ICE CRIM allele. Simultaneous disruption of multiple genes was also achieved when there are multiple sgRNA expression cassettes embedded within an activated ICE CRIM. Our mouse model can be used to generate an in vivo mutants pool for the in situ functional screen. I observed that various mutant alleles resulting from imprecise indel events can provide useful tags for in vivo clonal analysis, and the results suggested a monoclonal origin of hematopoietic neoplasms. The experimental results also indicated that DNA mismatch repair deficiency accelerates tumorigenesis in a Trp53 mutant genetic background. The use of CRISPR/Cas9 system allows genomic sequence-based, systematic experimental designs for various comprehensive cancer gene studies in the future.