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Researchers at the University of Illinois at Chicago are the first to describe why the CRISPR gene editing sometimes fails to work, and how the process can be made much more efficient.
CRISPR is a gene editing tool that allows scientists to cut out unwanted genes or genetic material from DNA, and sometimes add a desired sequence or genes. CRISPR uses an enzyme called Cas9 that acts like scissors to cut unwanted DNA. Once the cuts are made on either side of the DNA to be removed, the cell initiates the repair to stick both ends of the DNA strand together, or the cell dies.
In a study published in the journal Molecular Cell the researchers showed that when gene editing using CRISPR fails, which happens about 15 percent of the time, it is often due to the persistent binding of the Cas9 protein to the DNA of the cut site.
Lead author Bradley Merrill, an associate professor of biochemistry and molecular genetics at the UIC College of Medicine, says that before, researchers did not know why the process had failed so random
. sites where Cas9 was a "failure", he remained tied to the DNA strand and prevented the cell from initiating the repair process, "Merrill said. He also said that Merrill, a UIC graduate student, Ryan Clarke, and his colleagues also found that Cas9 would likely be ineffective in the future. genome sites where RNA polymerases – enzymes involved in gene activity – were not active. Further study revealed that guiding Cas9 to anneal just one of the strands constituting the double helix of DNA favored the interaction between Cas9 and RNA polymerase, helping to transform a Cas9 "failed" in an effective genome editor.
this selection of strands consistent for Cas9 during genome editing forced the RNA polymerases to collide with Cas9 so that Cas9 was removed from the DNA
"J & # 39; I was shocked to choose one strand of DNA rather than another, on the genome edition, "said Clarke, the lead author of the article. the mechanism behind this phenomenon helps us better understand how Cas9's interactions with the genome can cause some editing attempts to fail and that, when designing a genome editing experiment, we can use this understanding. "
Understanding is important for those of us who need a genome modification to work well in the laboratory and to make genome editing more efficient and effective. safer in future clinical uses, "said Merrill [19659005] .This process, the interaction between Cas9 and the DNA strand is now known to be "the speed limiting step," Merrill said. This means that it is the slowest part of the process; therefore, changes at this stage have the most potential to influence the overall duration of genome editing.
"If we can reduce the interaction time of Cas9 with the DNA strand, which we now know how to make with RNA polymerase, can use less of the enzyme and limit the exposure, "said Merrill." This means that we have more potential to limit side effects or side effects, which is vital for future therapies that can affect human patients. "
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