Development of a CRISPR/Cas9 System in Giardia lamblia.

• Main Authors: Zi-Qi Lin, 林子琦
• Other Authors: 孫錦虹
• Format: Others
• Language: zh-TW
• Published: 2017
• Online Access: http://ndltd.ncl.edu.tw/handle/w2de22

碩士 === 國立臺灣大學 === 微生物學研究所 === 105 === 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.