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UIC researchers show that the persistent binding of Cas9 to a double-strand break results in the failure of CRISPR genome editing about 15% of the time. When RNA polymerases collide with Cas9 in one direction (template orientation), they can dislodge Cas9 and increase the efficiency of genome editing CRDIT: Ryan Clarke et al.
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 off unwanted genes or genetic material from DNA, and sometimes add a 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. According to lead author Bradley Merrill, an badociate professor of biochemistry and molecular genetics at the UIC College of Medicine, researchers did not know up to now why the process had failed so much. 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 added that Merrill, a UIC graduate student, Ryan Clarke, and his colleagues also found that Cas9 would likely be ineffective at the end of the year. 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 editor of the genome
. this consistent selection of strands for Cas9 during genome editing forced the RNA polymerases to collide with Cas9 so that Cas9 was removed from the DNA
"I was shocked to choose one strand of DNA rather than another on the genome edition, "said Clarke, the main author of the article." Discover the mechanism behind this phenomenon helps us better understand how the Cas9 interactions with the genome can make some editing attempts fail and that, when designing a genome editing experiment, we can use this understanding for our benefit. "
Understanding is important for those of us who need to modify the genome to work well in the laboratory and to make genome editing more efficient and safer in the world. future clinical uses, "said Merrill [1965] 9007]. 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 greatest potential impact on the overall duration of genome editing.
"If we can reduce the interaction time of Cas9 with the DNA strand, which we now know to do 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 may affect human patients. "
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