麻豆淫院

Researchers at the Max Delbr眉ck Center have developed a new method to discover how DNA controls genes. Their technique, in Cell Genomics, can reveal the genetic "switches" that regulate important genes more quickly than existing methods.

Most of the human genome does not code for proteins. Instead, much of it consists of regulatory regions. Like switches that turn lights on and off, these regions of nucleotides鈥攃alled transcriptional enhancers鈥攄etermine where and when a gene is active, and largely control how much of the corresponding protein a cell produces.

Defects in the genetic code of such regulatory elements can cause developmental defects and disease. But compared to protein-coding regions, they are difficult to identify because they are often located far from the genes they regulate and lack a well-defined genetic code.

Scientists led by Dr. Dubravka Vu膷i膰evi膰 in the Computational and Regulatory Genomics lab of Professor Uwe Ohler have created a powerful new tool to uncover these regions that control our genes.

Called targeted single-cell activation screen (TESLA-seq), it combines CRISPR-based gene activation (CRISPRa)鈥攁 gene regulation technique that uses an engineered form of the CRISPR-Cas9 system to enhance the expression of specific genes鈥攁nd targeted single-cell RNA sequencing to identify regulatory regions more quickly and accurately than other methods.

"With this method, we can actually test how thousands of candidate regulatory elements in the genome are capable of switching genes on鈥攁nd find out exactly what genes they influence," says Vu膷i膰evi膰, lead author of the study.

Mapping regulatory elements

To showcase the technique, the study focused on a gene called PHOX2B, which is essential for nervous system development. Mutations in the gene have been linked to neuroblastoma, a cancer of nervous system tissue that primarily affects children.

They concentrated on a large region surrounding PHOX2B, designing 2鈥�3 guide RNAs (gRNAs) for each 100bp segment, or chunk of DNA, for a total of 46,722 gRNAs. This set probed the entire genomic landscape of the PHOX2B gene, as well as other neighboring genes, for potential roles as regulatory switches.

They then transferred each gRNA into a single human neuroblastoma cell. The gRNA told the CRISPRa system where to go and directed it to activate any regulatory regions that might be present in the "chunk." They identified more than 600 regions鈥攃alled CaREs (CRISPRa-responsive elements)鈥攖hat altered cell growth when activated.

The team then zoomed in on about 200 CaREs in more detail and used targeted single-cell RNA sequencing to read out both the gRNA inside each cell and the RNA expressed from nearby genes. This allowed them to link each CaRE to any of the over 70 genes in the PHOX2B region, whose expression changed in that cell. They also found direct connections between CaREs, important regulators of SHISA3 and APBB2, which are involved in cancer and Alzheimer's disease.

Surprisingly, many CaREs control genes far away, skipping over nearby genes entirely鈥攕omething other methods often miss.

"TESLA-seq doesn't just capture what's happening in one cell type, it can reveal potential connections between genes and regulatory regions across different biological systems," says Ohler.

This is significant because many diseases affect more than a single tissue type, adds Vu膷i膰evi膰.

"The technique can be used to study the vast, uncharted parts of our DNA that influence health and disease across multiple organ systems and can help us to design more precise and effective therapies."