利用CRISPR/Cas9與電穿孔技術產製基因嵌入小鼠模型

Abstract

CRISPR/Cas9系統源自於原核生物的後天免疫系統，近年來由於其能夠產生DNA雙股斷裂的特性，被發展並廣泛應用於基因編輯。然而，在利用CRISPR產製基因轉殖小鼠時，需利用耗費人力且費時的顯微注射將實驗材料遞送入原核胚。新近發展出了替代方法，例如電穿孔技術可取代技術門檻較高的顯微注射。此外，進行基因轉殖小鼠實驗需要使用大量的受精卵，若進行自然配種，會耗費較多公鼠及人力，且受精時間點不易控制。另外，針對如何提高大片段基因嵌入基因座的效率，仍舊是個待解決的問題。 本研究結合了CRISPR/Cas9系統與電穿孔技術，配合小鼠體外受精、凍卵與解凍技術，建立一套操作時間選擇較有彈性、耗費人力及時間較少且成功率較高的基因嵌入小鼠產製流程及實驗條件。首先，建立小鼠體外受精及受精卵冷凍與解凍系統，得到受精率約80%，解凍存活率約60%。另外，本研究測試利用電穿孔技術將EGFP mRNA送入受精卵中的效率。接著，利用不同電擊次數，決定出最佳的電穿孔實驗條件。同時在不同電擊次數條件下，針對單一切點產生的indel與大片段基因剔除的效率進行分析。最後，利用小片段基因嵌入實驗驗證電穿孔的最佳條件，同時決定最佳的模板濃度與設計策略。本研究也測試一種小片段互補寡去氧核醣核酸的策略，實驗結果顯示此策略可能提升基因嵌入效率的初步證據。綜合以上實驗，本研究已建立一套產製基因嵌入小鼠的最佳製程。

The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR associated protein 9 (Cas9) system, which discovered as a bacteria adaptive immune system at first, becomes a widely used genome editing tool owing to its ability to form a site specific DNA double stand break (DSB). However, when generating transgenic mice by CRISPR/Cas9 method, it is necessary to use a laborious and time-consuming microinjection to deliver experimental materials into pronuclear embryos. Recently, an alternative method has been developed. For example, electroporation technology can replace the higher technical threshold of microinjection. In addition, the experiment of transgene mice requires a large number of fertilized eggs. If natural mating is carried out, it will consume more male mouse and manpower, and the time of fertilization is difficult to control. Moreover, how to improve the efficiency of large fragment knock-in to gene locus is still a problem to be solved. This study combines CRISPR/Cas9 system and electroporation technology, combined with mouse in vitro fertilization, zygote freezing and thawing techniques to establish a set of knock-in mice production process and experimental conditions with flexible operation time, low labor and time, and high success rate. First, a mouse in vitro fertilization and zygote freezing and thawing system was established, and the fertilization rate was about 80%, and the thawing survival rate was about 60%. In addition, test the efficiency of electroporation by delivering EGFP mRNA into fertilized eggs. Next, using different numbers of shocks, to determine the optimal electroporation experimental conditions. At the same time, under the condition of different electric shock times, analyze the efficiency of indel and large segment gene knockout generated by single cutting site. Finally, use small-fragment knock-in experiments to verify the optimal conditions for electroporation, while determining the optimal template concentration and design strategy. This study also test a small fragment complementary ODN strategy. The experimental results show the prima facie evidence that this strategy may improve gene knock-in efficiency. Based on the above experiments, this study has established an optimal process for producing gene knock-in mice.