DNA is transcribed into RNA, which is then translated into proteins. This is the central dogma of molecular biology – a mantra taught to every biology undergraduate student to be recalled and expanded upon throughout our education. However, several cellular machines shepherd the RNA from their moment of birth on the DNA, to producing the necessary proteins, all the way to their eventual demise in the cytosol. These machines – collectively dubbed RNA regulators – are critical for proper gene expression and cellular health. While researchers understand some aspects of RNA regulation, we lack unbiased tools to query them in high throughput. To solve this problem, the Subramaniam Lab in the Basic Sciences Division has built a new genetic screening tool based on CRISPR genome editing.
During their lifetime inside the cells, each RNA interacts with thousands of proteins. These RNA-protein interactions can impact RNA processing, localization, translation, and stability. Their regulatory functions are crucial for proper protein synthesis, but interrogating the function of any particular RNA-associated protein is challenging. Traditionally, researchers have taken biochemical approaches to identify which binding proteins interact with any specific RNA. “You’re not necessarily measuring function; you’re looking for things that bind, but they don’t tell you which binding is actually functional,” notes Dr. Rasi Subramaniam, senior author of the study.
Given the nuances of RNA regulation, Subramaniam and former graduate student Dr. Patrick Nugent decided to take an untargeted genetic approach to identifying new regulators. To do this, they developed ReLiC, a high-throughput approach that combines CRISPR screening with RNA barcoding to identify regulators of RNA biology.
To start, the team integrated a gene encoding the Cas9 enzyme and a DNA library encoding guide RNAs targeting over 2000 known RNA-associated proteins into human cells. This results in a gene knockout directed by the specific guide RNA in each cell. Typically, in a CRISPR screen, researchers would interrogate the effect of each gene knockout by looking at cellular outcome like proliferation, survival, or protein levels. “The challenge is that if you see changes in protein level or cell survival, it doesn’t necessarily tell you what’s happening to a specific RNA,” highlights Subramaniam.
To fully capture changes to RNAs in a high-throughput way, the team linked the CRISPR guide RNA to an RNA-encoding gene with a unique barcode. “Once it goes into the cell and the cell transcribes these two regions, they float around differently. The guide is cutting DNA, and the [barcoded RNA] is…in the cytoplasm,” explains Subramaniam. If the team detects changes in processing or translation of their barcoded RNA, they can determine the gene causing these changes by cross referencing the barcode with its linked guide RNA.
Having the barcoded RNA associated with a specific gene knockout opens the door for a slew of functional assays interrogating regulatory changes to the barcoded RNA. Subramaniam and his team used many of these approaches to detect differences in splicing, translation, and degradation of their barcoded RNA. “[Combining CRISPR screens with RNA barcoding] suddenly opens up our ability to look at RNA biology that was essentially invisible,” says Subramaniam. While this tool was developed using a barcoded RNA without implications for human disease, Subramaniam hopes his group can leverage this tool to discover novel regulators of disease-associated RNAs.
This work was supported by funding from the National Institutes of Health and the National Science Foundation.
Fred Hutch/UW/Seattle Children’s Cancer Consortium members Drs. Stanley Lee, Andrew Hsieh, and Arvind Rasi Subramaniam contributed to this work.
Nugent PJ, Park H, Wladyka CL, Yelland JN, Sinha S, Chen KY, Bynum C, Quarterman G, Lee SC, Hsieh AC, Subramaniam AR. 2025. Decoding post-transcriptional regulatory networks by RNA-linked CRISPR screening in human cells. Nat Methods. 22(6):1237-1246. doi: 10.1038/s41592-025-02702-6.
Kelsey Woodruff is a PhD candidate in the Termini Lab at Fred Hutch Cancer Center. She studies how acute myeloid leukemia cells remodel the sugars on their membranes to reprogram cancer cell signaling. Originally from Indiana, she holds a bachelor's degree in Biochemistry from Ball State University. Outside of lab, you can find her crocheting and enjoying the Seattle summers.
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