[ad_1]
Stanford University researchers have revised the CRISPR-Cas9 gene editing technology to manipulate the genome in three-dimensional space, enabling them to transport genetic extracts to different locations in the nucleus of a cell.
The new technique, called the CRISPR genome organization or simply CRISPR-GO, uses a modified CRISPR protein to rearrange the genome in three dimensions. If CRISPR is like molecular scissors, CRISPR-GO is like molecular claws, capturing specific fragments of the genome and plunging them into new nucleus locations. But it is not just a physical relocation: moving genetic elements can change how they work.
The research sheds new light on how the spatial organization of the genome in the nucleus governs the function of the cell as a whole.
"The question of why space organization in a cell is important is important, and that's also not an issue that scientists are listening to," said Stanley Qi, PhD, assistant professor in bioengineering and in chemical biology and systems. "CRISPR-GO could be an opportunity to answer this question by allowing us to target, move and move very specific DNA segments, and see how their new locations in the kernel are changing their operation."
Most mammalian cells contain a nucleus that contains more than 6 feet of DNA, if elongated. This genetic material determines the fate of cells and, if moved or damaged, can lead to disease. Previous studies have shown that DNA tends to clump in certain areas of the nucleus. The impact of this placement on the function of DNA is however not yet clear.
In the proof-of-principle study, Qi studied CRISPR-GO in three distinct subregions of the nucleus, testing a global hypothesis: do genes and other genetic elements behave differently in different areas of the nucleus?
Until now, their data show that specific compartments and some protein bodies floating freely in the nucleus may influence the function of the repositioned DNA. Depending on the location of the genetic material, some nuclear regions repress the expression of genes and others accelerate telomere growth, and then cell division. A single protein body may even have the power to suppress the formation of tumors.
A study detailing this research will be published online on October 11 in Cell. Qi is the main author. Haifeng Wang, postdoctoral researcher, is the lead author.
Bridging the gap
Demystifying the physical details of the genome has proven to be a tedious task, but some existing technologies allow scientists to scan the cells and see how their guts are physically organized. What is missing is a way to alter this organization. CRISPR-GO is the first to offer researchers a way to do this.
By disabling the "clipping" mechanism of CRISPR-Cas9, the editing tool becomes more of a delivery system, used by Qi to deliver small portions of DNA via a programmable guide ARN. to a new location in the kernel.
CRISPR-GO has three essential parts. First, there is what Qi calls "the address" of the genetic target that you want to move, a segment of DNA targeted by a complementary strand of binding RNA. Then you need the destination address – the specific part of the DNA of a nuclear compartment to which you want to move the chromatin. Finally, there is the "bridge", which in this case is a catalyst that causes freezing of the target DNA in its new focus in the nucleus.
"Children often like to build small railroad tracks to help trains pass from one station to another," Qi said. "It's not so different from what we do here."
Different room, different function
Qi describes the features of nuclear compartments as spaces of a home. In every room of your house, you do different things – in the kitchen, you cook; in the bedroom, you sleep. In the kernel of a cell, the same concept applies. The nucleus has multiple compartments that all play a specific role in maintaining the overall functionality of the cells. Qi and his lab investigated three distinct areas of the nucleus, trying to determine if they could change chromatin function as a function of their displacement.
Using CRISPR-GO, the researchers observed that genes displaced in a part of the nucleus called Cajal's body, an amorphous and somewhat mysterious drop of proteins and RNA, stopped expressing proteins.
"We were very excited to see this – it's the first time that researchers have evidence that the cajal body can have a direct effect on gene regulation, repressing in this case the expression of genes." Qi said. "This suggests that the cajal body has an unexpected role in controlling transcription." This could be huge because transcription is an important process that synthesizes the "code" for protein production.
When Qi used CRISPR-GO to move DNA from telomeres – the molecular capsules of chromosomes associated with longevity – from the middle to the edge of the nucleus, telomeres stopped growing, interrupting the cell cycle and reducing cell viability . The reverse, however, was produced when the telomeres were brought closer to the body of Cajal: they developed and, in doing so, increased cell viability.
The third application used CRISPR-GO to form a promyelocytic leukemia body. This protein globule is known to suppress pro-tumor genes. By placing it next to the carcinogenic genes in the nucleus, Qi plans to check if it can help curb the formation of tumors.
"Another unique advantage of CRISPR-GO is that we can track the interactions between chromatin DNA and nuclear compartments in real time under the microscope," said Wang.
While the evidence presented by CRISPR-GO is interesting, the research is still at a pilot stage and much more needs to be done before the results can be confirmed, Qi said.
"We are very excited about the potential here and, even though we answered a few questions, we opened about twenty others," Qi said.
It will be even more important to understand why these localized effects occur in specific nuclear compartments and what is the underlying cause, he said. One day, Qi hopes that this line of research will have repercussions on human health.
Source link
