建立DNA錯配修復功能缺失的ICE CRIM小鼠模式

Abstract

癌症為國人十大死因之首，癌症基因體也是學術研究的重點之一。在細胞中，一般的代謝活動和環境因素都能造成DNA損傷，這些損傷有可能造成DNA的破壞，改變基因編碼的內容，或是引發在細胞基因體中的潛在有害突變，進而造成基因體不穩定性，大幅增加細胞癌化的可能。因此當細胞產生DNA損傷時，DNA就會啟動自我修復機制。DNA 修復能力對生物體維持基因體的完整性是至關重要的。我們想要針對抑癌基因(tumor suppressor genes)中的DNA修復基因(DNA repair genes) 進行相關研究。Mlh1, Msh2 這兩個基因，是參與DNA 錯配修復(mismatch repair) 最重要的兩個角色，其主要功能在修復DNA複製過程中所產生的鹼基-鹼基錯配。我們想藉由近年來興起CRISPR/Cas9 的基因體編輯技術，在小鼠活體內進行Mlh1, Msh2的破壞來研究其分子間之遺傳交互作用與癌症進程的相關性。CRISPR/Cas9的基因體編輯技術，可以讓我們在不必經由冗長、費力的動物交配、繁殖之下，於活體內同時針對單個或多個基因製造突變的體細胞群。我們想建立一個攜帶著可藉由組織特異性環化重組酶(Cre)啟動的iCas9、帶有毛色標幟的、並針對Mlh1,Msh2進行破壞的sgRNA的複合轉基因sgRNA-iCas9小鼠模式，稱為ICE CRIM(Inducible Cas9 Effector/ CRIspr Mutagen)。這個小鼠模式將可以搭配不同組織特異性重組酶(Cre)小鼠，進而條件式的啟動Cas9的活化。在小鼠體內特定組織進行Mlh1,Msh2的破壞，提供一個Mlh1,Msh2突變異質性高、易發癌症的背景，未來可能讓我們得以在突變的體細胞群上進行正向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。

Cancer is the leading cause of mortality worldwide. Cancer genomics is a hot topic for biomedical research. The process a cell changes from a normal somatic cell into a cancer cell involves many factors. Genome instability and the accumulation of mutations are highly correlated to the incidence of cancer. Many studies found that oncogenes and tumor suppressor genes are directly related to cancer. Here I want to focus on the DNA repair genes, a small group of tumor suppressor genes. Cells in human body receive tens of thousands of DNA lesions per day. When a cell suffered from DNA damage, the cell will start self-repair mechanisms, so that most of the mutations can be restored. DNA repair pathways can therefore reduce the mutation rate and safeguard the integrity of genome. One of the DNA repair mechanisms is DNA mismatch repair (MMR). The MMR machinery recognize and repair base-base mismatches produced during DNA replication. Mlh1 and Msh2 are two major players in DNA mismatch repair. Here I would like to generate a mouse model deficient in DNA mismatch repair by destroying Mlh1 and Msh2 simultaneously. I used the latest CRISPR/Cas9 gene editing technology to perform somatic mutagenesis of target gene(s) in vivo. We can get single or multiple genes mutated with minimum animal breeding efforts. It is also possible to investigate the relationship between genes and human diseases by manipulating gene editing in animals. I would like to generate a coat-color iii tagged transgenic mouse carrying an inducible Cas9 effector (ICE) cassette, which is activable by Cre, and CRIspr Mutagens (CRIM), the sgRNAs target either Mlh1 or Msh2. Mating with Cre transgenic mouse, Cas9 can be conditionally activated, and Mlh1 and Msh2 will be destroyed in different tissues in an ICE CRIM mouse. In the future, our model could potentially provide a ‘mutator phenotype’ background caused by Mlh1 and Msh2 heterogeneous mutations, allowing us to discover the genetic interactions between different cancer gene mutation events for cancer initiation and evolution in the mutant somatic cells.