Understanding the meaning of the human genome codes is one of the keys to unlock the secrets of life. Despite having a fairly good grasp of the sequences of the human genome, we are still far from... Show moreUnderstanding the meaning of the human genome codes is one of the keys to unlock the secrets of life. Despite having a fairly good grasp of the sequences of the human genome, we are still far from understanding the functions of most parts of the genome and their involvement in diseases. The application of the CRISPR-Cas9 genome-editing systems revolutionized the way to study the function of the genome, not only the coding genes but also the non-coding genome. In this thesis, multiple CRISPR screening systems were designed and used to study the transport of chemotherapeutic drugs and the functions of non-coding regulatory elements in distinct biological pathways. Using the CRISPR screening systems, we identified previously known drug exporters such as ABCB1 and ABCG2 and a new doxorubicin importer gene SLC2A3 (GLUT3). We used an innovative dual-CRISPR system to study the functions of noncoding regulatory elements (NCREs) in their endogenous genomic environment. We provide the broad research community with a new tool to study the functions of NCREs in different biological aspects, and it is expected many more important NCREs will be discovered in the future, which would not only be relevant to fundamental biology but also to human diseases. Show less
PurposeTissue-specific drug uptake has not been well studied, compared to the deeper understanding of drug resistance mediated by the cellular efflux system such as MDR1 proteins. It has been... Show morePurposeTissue-specific drug uptake has not been well studied, compared to the deeper understanding of drug resistance mediated by the cellular efflux system such as MDR1 proteins. It has been suggested that many drugs need active or defined transporters to pass the cell membrane. In contrast to efflux components induced after anti-cancer drugs reach the intracellular compartment, drug importers are required for initial drug responses. Furthermore, tissue-specific uptake of anti-cancer drugs may directly impact the side effects of many drugs when they accumulate in healthy tissues. Therefore, linking anti-cancer drugs to their respective drug import transporters would directly help to predict drug responses, whilst minimizing side effects.MethodsTo identify drug transporters of the commonly used anti-cancer drug doxorubicin, we performed focused CRISPR activation and knockout genetic screens targeting all potential membrane-associated transporters and proteins. We monitored the direct uptake of doxorubicin by fluorescence-activated cell sorting (FACS) as the screening readout for identifying transporters/proteins directly involved in doxorubicin uptake.ResultsIntegrating the data from these comprehensive CRISPR screenings, we confirmed previously indicated doxorubicin exporters such as ABCB1 and ABCG2 genes, and identified novel doxorubicin importer gene SLC2A3 (GLUT3). Upregulation of SLC2A3 led to higher doxorubicin uptake and better cell killing, indicating SLC2A3 could be a new marker to predict doxorubicin drug response and minimize side effects for the personalized application of this conventional chemotherapeutic drug.ConclusionsOur study provides a comprehensive way for identifying drug transporters, as exemplified by the commonly used anti-cancer drug doxorubicin. The newly identified importers may have direct clinical implications for the personalized application of doxorubicin in treating distinct tumors. Our results also highlight the necessity of combining both CRISPR knockout and CRISPR activation genetic screens to identify drug transporters. Show less
