CRISPR Reveals Hidden Functions of Noncoding RNA

Genes contain instructions for protein production, and the central dogma of biology states that this information flows from DNA to RNA to proteins. However, only about 2% of the human genome actually codes for proteins, and the role of the remaining 98% is largely unknown.

A key question in human genetics is understanding whether these regions have any function. Historically, some have even labeled these areas as "junk."

Now, a new study in Cell reveals that some noncoding RNAs are not merely "junk" but are functional and play significant roles in cellular processes, including cancer and human development.

Researchers at New York University and the New York Genome Center used CRISPR technology targeting RNA instead of DNA to screen the genome, identifying nearly 800 noncoding RNAs essential for the function of diverse human cells across various tissues.

This survey of functional noncoding RNAs advances our understanding of the human genome. It demonstrates the potential of CRISPR screens that specifically target RNA—even those that don’t code for proteins.”

Neville Sanjana, Associate Professor of Biology, New York University

Neville Sanjana is also an Associate Professor of Neuroscience and Physiology at NYU Grossman School of Medicine, a core faculty member at the New York Genome Center, and the study’s senior author.

A More Precise CRISPR for RNA

The CRISPR gene-editing technology has transformed biomedical research, with applications ranging from boosting crop yields to treating blood disorders by editing DNA in blood cells.

Most CRISPR methods utilize the enzyme Cas9 to edit DNA. However, a newer approach employs the enzyme Cas13 to more precisely target RNA, avoiding disruption of nearby protein-coding genes and regulatory elements. Sanjana’s lab previously showed that a CRISPR-Cas13 platform targeting RNA can be optimized to screen the entire transcriptome, the collection of genetic information transcribed into RNA molecules.

While many studies have used sequencing to measure RNA expression, it has been challenging to determine whether specific RNA molecules are essential for cell function.

“We now have this technology, but the biological question remains: which parts of the noncoding genome are actually functional?” asked Simon Müller, the co-first author of the Cell study and a Postdoctoral Associate in Sanjana’s lab.

Not Junk After All

Using CRISPR-Cas13 to edit RNA and minimize off-target effects, the researchers systematically profiled nearly 6,200 gene pairs of long noncoding RNAs (lncRNAs) and adjacent protein-coding genes across five human cell lines, including kidney, leukemia, and breast cancer cells.

They employed CRISPR to perturb or knock down each lncRNA, observe the outcomes—whether the cell dies, ceases to proliferate, or can tolerate the change—and determine the essentiality of each lncRNA.

With Cas13, we can specifically ask, ‘What are the functions of those transcripts?’ They are not junk—we found that they are really important and essential to cells growing and dividing.”

Wen-Wei Liang, Study Co-First Author Postdoctoral Associate, New York University

The researchers identified 778 essential lncRNAs, including a core group of 46 that are universally essential and 732 that have functions specific to particular cell types.

They then compared these essential lncRNAs to protein-coding genes. For protein-coding genes, if a gene was critical to one of the five cell lines, it was likely necessary in the others. In contrast, essential lncRNAs were more specific to particular cell types.

The researchers also explored whether essential lncRNAs influence nearby protein-coding genes, a question that had yet to be previously investigated for noncoding RNAs. They found that the vast majority of crucial lncRNAs function independently of the nearest protein-coding genes.

The team also discovered that essential lncRNAs regulate key pathways involved in cell proliferation, a process critical for both human development and cancer. Their loss can disrupt cell progression and induce cell death. Notably, many essential lncRNAs were highly expressed in tissues during early human development, with expression levels decreasing in later stages, suggesting a significant role for certain lncRNAs during development.

Additionally, in an analysis of approximately 9,000 tumors, the researchers identified lncRNAs with altered expression in specific tumor types. They pinpointed those whose expression levels were linked to better or worse survival outcomes in various cancers.

These noncoding RNAs may yield new biomarkers and therapeutic targets for cancer treatment, a potential opportunity for personalized medicine given their cell type-specific expression.”

Neville Sanjana, Associate Professor of Biology, New York University