My Project Outline: "Characterizing the SON Gene via CRISPR-Mediated Protein Tags"
Title:
“Characterizing the SON Gene via CRISPR-Mediated Protein Tags”
Abstract:
Recent research has indicated that the spatial organization of the nucleus plays a critical role in gene expression and disease pathology. Yet the fundamental logic underlying this process is still poorly understood. A number of membraneless organelles within the nucleus are thought to be involved in gene expression, working together in a complex assembly to conduct functions like splicing and transcription.
One particular type of substructure in this category– those being nuclear speckles –appear to be integral to the organization of the aforementioned nuclear architecture. The SON protein is now widely thought to be central to this architecture, being a critical participant in the RNA splicing process (wherein immature RNA has unneeded sections removed before the final transcript is translated into protein form). However, its in vivo dynamics and functional contributions are still largely uncharacterized.
This project aims to directly visualize the activity of SON protein in live HepG2 cells. This goal will be achieved by tagging the SON locus with a fluorescent HaloTag protein, through a CRISPR/Cas9-mediated knock-in. Single-guide RNA molecules (“sgRNAs”) will target complementary sites near the SON gene; concurrently, homology-directed repair (HDR) templates containing the HaloTag sequence will be spliced into the targeted region. Two separate HDR templates will be used, respectively using microhomology-mediated and nonhomologous end-joining DNA repair techniques (MMEJ and NHEJ).
Successfully modified cells will be selected via puromycin treatment and genetic sequencing. The primary functional analysis of modified cells will be fluorescence microscopy, with the goal of investigating the localization patterns of SON protein through in vivo examination. If time permits, a secondary aim is to implement a protein tag degradation system, investigating the functional necessity of SON in maintaining nuclear speckle integrity. This experiment’s conclusions may ultimately contribute to our scientific understanding of the relationships between nuclear architecture and gene expression patterns.
Research Question: “What is the role of SON protein in organizing and regulating the activity of membraneless nuclear organelles?”
Background:
Eukaryotic gene expression– the process through which information in the genome is used to generate RNA transcripts, and ultimately functional proteins –is heavily reliant on the precise coordination of transcription and pre-mRNA splicing. This complex interplay is governed by the spliceosome; this is a dynamic complex of ribonucleoproteins which assemble to remove unwanted regions (introns) and connect the desired regions (exons) in immature RNA transcripts (Sharma et al., 2011). Among the membraneless organelles that cooperate to form the spliceosome are the nuclear speckles, irregular domains enriched in spliceosome motifs like SR proteins and long noncoding RNAs.
Research has shown these speckles to behave as central hubs coordinating multiple phases of gene expression: storage and modification of splicing factors, recruitment of said factors to transcription sites, and organization of pre-mRNA processing (Morimoto and Boerkoel, 2013). SON protein, according to recent findings, is one of the primary scaffolding proteins maintaining the structure of these nuclear speckles. Defects in or depletion of SON have resulted in chaotic dispersion of speckle proteins, impaired localization of splicing factors, and other related defects in the splicing process (Lu et al., 2014).
While some research has been done in recent years on the precise relationship between SON and gene expression, much of this research has made use of methodologies like plasmid overexpression– methods which may provide a less accurate picture of natural protein function (Roberts et al., 2017). Labeling SON with an endogenous protein tag via CRISPR editing provides a higher-fidelity method of observing SON’s localization and functionality over time. Additionally, if the inducible degradation arm of this project is to be pursued, that would be an opportunity to further evaluate SON’s necessity for the integrity and functionality of nuclear speckle proteins.
Works cited:
Morimoto M, Boerkoel CF. “The role of nuclear bodies in gene expression and disease”. Biology (Basel). 2013 Jul 9;2(3):976-1033. doi: 10.3390/biology2030976.
Lu X, Ng HH, et al. “The Role of SON in Splicing, Development, and Disease.” Wiley Interdisciplinary Reviews: RNA, vol. 5, no. 5, 2014.
Roberts B, Haupt A, et al. (2017). “Systematic gene tagging using CRISPR/Cas9 in human stem cells to illuminate cell organization”. MBoC. https://doi.org/10.1101/123042
Sharma A, Takata H, et al. "Son maintains accurate splicing for a subset of human pre-mRNAs". J Cell Sci. 2011 Dec 15;124(Pt 24):4286–4298. doi:10.1242/jcs.087775.
Methodology:
Note: Methodology may be subject to minor changes depending on time and resources available; mentors will be consulted as necessary.
- Split HepG2 cells from stock culture; begin basic maintenance procedures (replenishing medium, passaging periodically, etc.)
- Thaw prepared plasmid mixtures containing Cas9 as well as cloned sgRNAs and donor oligonucleotides; these mixtures should correspond with the NHEJ and MMEJ repair mechanisms respectively.
- Send plasmid mixtures for Plasmidsaurus sequencing; compare to reference sequences on Benchling database to confirm components are correctly-identified and ready for splicing.
- Split cultured HepG2 cells; transfect with sgRNA/oligo mixture via lipofectamine procedure.
- Check for successful transfection via puromycin screening; sequence to confirm success, and expand clones in new dishes for further investigation.
- Perform fluorescence microscopy on transfectants, investigating localization patterns of GFP-tagged SON protein. Optionally (depending on time available), perform additional knockout analysis based on protein tag degradation of modified SON protein.
Potential Impact:
The exact physical dynamics of nuclear speckles, and their precise functions as regards transcriptional regulation, continue to evade scientific understanding; this project cannot singlehandedly illuminate these unknown areas. However, the methodology and conclusions of this experiment could serve to substantively inform future research into the questions of subnuclear organization. The protein tagging strategy I’m employing provides a model method of investigating the architecture of nuclear organelles with minimal disruption to natural cell physiology. The repercussions of employing the DNA repair strategies of MMEJ and NHEJ in parallel could facilitate many future CRISPR-splicing-based experiments. And the possible extension of this project focusing on SON knockout through protein-tag degradation (which I will continue to research in this lab, regardless of whether or not I have the time to during this summer), could shed light on the unexplored role of SON protein in RNA splicing, and perhaps transcriptional regulation as a whole.
Resources & Support Needed:
Throughout this whole process, I’ll be relying on the feedback of a number of mentors and fellow researchers to ensure I’m applying newly-learned skills correctly (principally my PI, Dr. Sreejith Nair, and my postdoctoral colleague, Dr. Shravan Girada). In terms of physical materials I’m relying on, I’ll need to acquire pre-prepared CRISPR templates from my colleague Xinrui Wei, as well as viable HepG2 cells for transfection purposes from my colleague Weiyi Guo.
Hi, everyone! I'm Martin. I'm a third-year student at Georgetown majoring in Biology (with a concentration in Molecular Bio), and I'm planning to minor in Japanese. My focus has always been on cancer biology; so many people I've known have been affected by it, and my life's goal is to contribute to a cure. I'm half-American, half-Bulgarian, the child of two diplomats; I speak English fluently, and intermediate Bulgarian, Japanese, and Spanish. I have a lot of different interests outside of science: basketball and baseball, chess, politics, music, and a whole other list that's way too long for this blurb. Here's a random fact about me which I think is kinda cool: I once saw the tallest manmade thing on Earth (the Burj Khalifa) and the tallest thing on Earth, period (Mt. Everest) from the window of the same flight!
My research project for Laidlaw this summer is an application of the fields I've spent so much time studying- the focus is on cancer genetics, molecular biology, and laboratory technique. The goal of this project is to uncover information about a certain protein called SON, which is thought to be involved in the processes of gene splicing and transcription into RNA. Errors in these functions often result in cancer, so figuring out the roles and behaviors of proteins like SON could really help future endeavors like genetic libraries and drug discovery programs. I won't bog this down with an excessively detailed explanation of my method, but in brief, I'm using CRISPR technology to edit the SON gene and add DNA which encodes something called a protein tag. CRISPR can use a few different DNA repair techniques as a sort of "glue" to insert this protein tag; I'll be testing two of these techniques (NHEJ and MMEJ) to add depth to the experimental process. When the modified gene gets translated into SON protein, this tag gets produced as well. The tag can be lit up and investigated with a fluorescent microscope to determine where in the cell the SON protein localizes; it can also be targeted for degradation, allowing me to research the behaviors of cells with no SON protein and thus deduce its function through this knockout treatment. Which of these goals I can accomplish depends on the time and resources I have available, but I hope that summary sheds light on the problems I'm trying to tackle with this research project. In a word, I want to find out what SON does, and I'm going to use CRISPR-mediated protein tags to find out.
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