建立梨形鞭毛蟲的CRISPR/Cas9系統

Abstract

梨形鞭毛蟲(Giardia lamblia)是全球性之致病性的腸內原蟲寄生蟲，感染途徑是透過飲用受汙染之水源。滋養體寄生於小腸，隨著腸內環境改變會進行囊體化作用(encystation)，形成具有囊壁之感染型 ── 囊體(cyst)。囊壁是梨形鞭毛蟲的構造，能夠抵禦惡劣環境下生存所需，其主要由多醣類還有蛋白質所構成。梨形鞭毛蟲的生活史分成兩種型態，一種為擁有兩個核並具有鞭毛運動性之滋養體(trophozoite)，另一種是四個核的成熟囊體。在囊體化階段中，囊體蛋白質(cyst wall protein, cwp)的表現量會大量增加。 近年來，基因组编輯技術(genome editing technology)透過特定的核酸酶在DNA上進行剪切。基因剪切後進一步透過兩種方式來達到修復動作，一則是非同源末端接合機制(non-homologous end joining, NHEJ)來修復DNA斷裂的切位，另一則是利用同源重组機制(homologous recombination)針對斷點附近的基因序列進行同源序列修復(homology direct repair,HDR)，而達到基因置換的結果。Cas9蛋白質會辨識入侵病毒或是外來質體上的Protospacer adjacent motif (PAM)，並且切割緊鄰的雙股genomic DNA區間，造成genomic DNA形成雙股斷裂，隨後進行基因修復。 過去沒有實驗室建立梨形鞭毛蟲的CRISPR/Cas9系統，本研究將建立一套梨形鞭毛蟲的CRISPR/Cas9系統，以利未來可以研究基因相關功能。針對myeloid leukemia factor (MLF) protein (orf 16424)基因來設計基因剔除系統。本實驗室發現MLF已知會出現在囊泡中，MLF大量表現時，梨形鞭毛蟲的囊壁蛋白質cwp1表現量會增加。將含有所需序列之DNA“修復模板’’、guide RNA和Cas9的質體透過轉染輸送到細胞內。DNA“修復模板’’內含有置換目標基因MLF基因上，下游基因序列和Puromycin resistance (Puromycin N-acetyltransferase, pac) gene，透過Cas9 蛋白質會誘導DNA斷裂，當細胞若進行HDR修復，就會將所設計好的質體和genomic DNA置換，使得細胞含有抗藥性基因。利用加入Puromycin來篩選具抗藥性的細胞，可成功篩選出行使HDR的細胞。利用PCR將基因放大做定序，也成功確認梨形鞭毛蟲MLF基因有部分成功被置換掉。本實驗也為了釐清MLF基因基因被置換掉的比率，透過即時定量聚合酶鍊式反應(quantitative PCR；qPCR)偵測genomic DNA置換比率。可以觀察到genomic DNA上的目標基因MLF部分剔除。在MLF部分基因剔除下，觀察相關蛋白質表現量和RNA表現量，可以發現成功剔除MLF部分genomic DNA下，MLF的RNA和蛋白質的量值也下降。透過螢光顯微鏡觀察型態方式觀察MLF囊泡數目量值下降，而MLF的調控目標-囊壁蛋白質cwp1蛋白質和RNA量值也下降。所形成的囊壁有變薄的趨勢。囊體計數下，可以觀察到形成的囊體數目也有下降。透過諸多證明CRISPR/Cas9系統是可以成功置換掉梨形鞭毛蟲的genomic DNA，也發現MLF對於cwp1有正調控。本實驗也將藥物去除，使得細胞質體脫落，來觀察失去藥篩選還有失去質體下的細胞和對照組的差別，結果和用藥物篩選時的表現量趨勢相同。 根據過去文獻，已知細胞在DSB (double strand break) 情況下，利用HDR修復的效率通常較低，即便是在表達Cas9、guide RNA和外源性修復模板細胞。由於Cas9蛋白質誘導DNA斷裂的效率相對較高和HDR的低效率，會促使細胞較會透過NHEJ的機制來修復。因此，本研究還透過NHEJ抑制劑─SCR7，會在修復時期抑制DSB的連接，並且阻止連接酶IV接合在DSB的DNA上，而導致在細胞內的DNA雙鏈斷裂的累積。SCR7還會抑制細胞內的NHEJ對連接酶IV依賴性，隨之激活內在凋亡途徑，提高HDR效能。故本實驗在轉染後24小時加入SCR7，用來抑制NHEJ參於修復機制的比率，提高HDR的效率。結果發現可以獲得較高的置換效能，相較於未使用SCR7的組別，MLF基因剔除比率提高，MLF的RNA和蛋白質的量值也下降。而MLF的調控目標-囊壁蛋白質cwp1蛋白質和RNA量值也下降。 另外也建立另一個CRISPR/Cas9系統，是利用neomycin來篩選出成功MLF基因，結果發現可成功剔除MLF部分genomic DNA下，MLF的RNA和蛋白質的量值也下降。而MLF的調控目標-囊壁蛋白質cwp1蛋白質和RNA量值也下降。囊體計數下，可以觀察到形成的囊體數目也有下降。本實驗也將成功基因置換成功的細胞株去藥後，去比對WB細胞株的相關基因、蛋白質的表現，也能夠再次確認細胞株有成功個別剔除myeloid leukemia factor (MLF) protein (orf 16424)基因。另外，還改變了DNA修復模板的形式為雙股模板，還有透過另一種方式將DNA“修復模板’’、guide RNA和Cas9整合在一條質體的方式，結果也是可以成功剔除MLF部分genomic DNA。結論是利用DNA修復模板的形式為雙股模板，是可以使質體脫去的效率提升，降低時間，可以比較和野生株的表現量。若是形成持續表現質體的單一質體的方式可以使質體一直持續性的表現Cas9、guide RNA和修復模板。也實驗利用neomycin來篩選出成功的細胞在轉染帶有MLF表現的質體，觀察MLF表現量也都有上升的趨勢。 由於CRISPR/Cas9系統建立，但不知是否對於其他基因也有效能。所以也針對HTH domain(orf2732)的基因做相同的設計，也發現可以達到部分剔除HTH domain基因，也可以發現當HTH domain基因目標基因部分剔除時，HTH domain的RNA和蛋白質的量值也下降。而HTH domain的調控目標-囊壁蛋白質cwp1蛋白質和RNA量值也下降。形成的囊壁也有變薄的趨勢。故透過此系統也可以剔除部分HTH domain基因。透過這個系統的建立，以利後面對於梨形鞭毛蟲的基因分析有極大的幫助。

Giardia lamblia is one of the most common protozoan parasites causing waterborne intestinal infections in humans. Cyst surives in hostile environment. G. lamblia have two stages in the life cycle: a flagellated trophozoite with 2 nuclei and an inert cyst with 4 nuclei. During encystation, differentiation from a trophozoite into a cyst, the cyst wall protein (CWPs) is highly synthesized in a concerted manner. Precise genome-editing relies on the repair of sequence-specific nuclease-induced DNA nicking or double-strand breaks (DSBs) by homology-directed repair (HDR) and (non-homologous end joining, NHEJ). The RNA guided enzyme Cas9, which originates from the CRISPR-Cas adaptive bacterial immune system, is transforming biology by providing a genome engineering tool. Recently, the CRISPR/Cas9 system was adapted for targeted genome editing in a variety of organisms. The Cas9 enzyme generates breaks in double-stranded DNA by using its two catalytic centers to cleave each strand of a DNA target site next to a Protospacer adjacent motif (PAM) sequence. After double-stranded DNA breaks are generated, DNA repair occurs . In this research, we developed a CRISPR / Cas9 system to remove the myeloid leukemia factor (MLF) protein (orf 16424) gene in G. lamblia. MLF has been found to induce cwp1 expression. We transfected the plasmid consisting of DNA 'repair template',guide RNA, Cas9 into the cells. The DNA 'repair template' contains upstream and downstream of MLF gene sequences with puromycin resistance gene, puromycin N-acetyl-transferase (pac). The cas9 gene product can induce DNA breaks. If the cells perform the repair action of HDR, the designed repair template will replace Giardia lamblia genomic DNA and make the cells contain drug-resistant genes.We used puromycin to select the drug resistant cells. We also used PCR to amplify the genes for sequencing and successfully confirmed that the MLF gene was successfully replaced. In order to clarify the rate of MLF gene replacement, we detected the replacement ratio by quantitative PCR (qPCR).We also measured the amount of expression of the relevant proteins and RNA expression through Western blot, RT-PCR, quantitative RT-PCR (qRT-PCR) and cyst count. Through above experiments we confirmed the genome replacement. It was observed that the target gene MLF was partially excluded from genomic DNA. In the MLF gene exclusion, it can be found that in the MLF part of the genomic DNA, MLF RNA and protein values also decreased. The amount of MLF vesicles was decreased by fluorescence microscopy, while the amount of protein and RNA of cystic protein cwp1 was also decreased. The formation of the wall has a thinning trend. The number of cysts formed can also be observed to decrease. Through a number of proven CRISPR / Cas9 system is able to successfully replace the Giardia genomic DNA. Also we found that MLF can positively regulate cwp1 expression. In this study, the puromycin was also removed, leaving the plasmid off, to observe effect of the loss of drug selection and loss of plasmid. According to the previous study, Cas9-mediated modification of the murine genome through NHEJ can reach efficiencies of 20–60%. But HDR has lower efficiency. Because NHEJ is error-prone and introduces unpredictable patterns of deletions, it is suitable for introducing small random mutations. HDR is less frequent than NHEJ and occurs only during S and G2 phase, whereas NHEJ occurs throughout the cell cycle. Scr7 targets the DNA binding domain of DNA ligase IV and reduces its affinity for DSBs and inhibiting its function. We used Scr7 to enhance the frequency of HDR by transiently blocking NHEJ, resulting in precise genome editing by CRISPR-Cas9. The MLF gene knockout ratio was increased, and the amount of MLF RNA and protein was also decreased compared to the group without the use of SCR7. In addition, another CRISPR / Cas9 system was established, and neomycin was used to screen out the successful MLF gene. The results showed that the MLF RNA and protein were also decreased in the MLF genomic DNA. While the MLF and cwp1 protein and RNA values also decreased. The number of cyst formed can also be observed to decrease. We will also succeed in isolating myeloid leukemia factor (MLF) protein (orf 16424) by successively demonstrating that the cell lines have succeeded in isolating the genes and proteins of the WB cell line after successful drug replacement. In addition, the DNA repair template has been changed in the form of a double strand template can remove target gene. Also the DNA 'repair template', guide RNA and Cas9 are integrated in a plasmid form can remove target gene. . The conclusion is that the use of DNA repair template in the form of a double strand template is the ability to make the removal of the plasmids to improve the efficiency, reduce the time, can be compared with the wild type of the amount of performance.If the formation of a single plastid can continue performance of Cas9, guide RNA and repair template. Also use of neomycin to screen out the success of cells in the transfection of MLF with the performance of the plasmids, observed MLF performance also increased. Since the CRISPR / Cas9 system was established, it was not known whether it was effective for other genes. Therefore, the HTH domain gene (orf2732) is also designed for knock down experiment. It is also found that the HTH domain gene can be partially excluded. It can also be found that the amount of RNA and protein of HTH domain also decreases when the target gene of HTH domain gene is partially eliminated. And HTH domain regulation target cwp1 protein and RNA values also decreased. The formation of the wall also has a thinning trend. So through this system can also be removed part of the HTH domain gene. Through the establishment of this system, it is of great help facilitate the subsequent analysis of the genetic analysis of Giardia.