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 이성욱 ( 2017-09-30 00:33:44 , Hit : 1988
 To Speed CRISPR/Cas9’s Search and Snip, Try Extra Cas9 and gRNA

http://www.genengnews.com/gen-news-highlights/to-speed-crispr-cas9-s-search-and-snip-try-extra-cas9-and-grna/81254994?utm_medium=newsletter&utm_source=GEN+Daily+News+Highlights&utm_content=02&utm_campaign=GEN+Daily+News+Highlights_20170929


GEN News Highlights
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September 29, 2017

The CRISPR/Cas9 gene-editing system is advantaged by its targeting mechanism’s inherent flexibility, but disadvantaged by this mechanism’s inherent slowness. The mechanism, which depends on a guide RNA (gRNA)-programmable protein, can take a long time to comb through a genome before it finally chances upon a target DNA sequence—up to six hours in a bacterial cell. The problem, say scientists based at Uppsala University, is slow kinetics. One way to overcome this problem, the scientists suggest, would be to use high concentrations of gRNA and Cas9.

This rate-enhancing possibility for the CRISPR/Cas9 system was raised in a study that appeared September 29 in the journal Science, in an article entitled “Kinetics of dCas9 Target Search in Escherichia coli.” This article explains that Cas9 must unwind the DNA at each location that it searches.

“Here we study the search mechanisms of the catalytically inactive Cas9 (dCas9) in living Escherichia coli by combining single-molecule fluorescence microscopy and bulk restriction-protection assays,” wrote the article’s authors. “We find that it takes a single fluorescently labeled dCas9 6 hours to find the correct target sequence, which implies that each potential target is bound for less than 30 milliseconds.”

Essentially, the researchers used two methods to measure how long Cas9 takes to find its target sequence. The first method showed that it takes as long as six hours for Cas9 to search a bacterium, that is, through four million base pairs. This somewhat unlikely result was also verifiable by means of the second, independent technique. The time found tallied with the number of milliseconds Cas9 has available for testing every position, which the researchers were able to measure by following labeled Cas9 molecules in real time.

“The results show that the price Cas9 pays for its flexibility is time,” said Johan Elf, Ph.D., the study’s senior author. “To find the target faster, more Cas9 molecules searching for the same DNA sequence are needed.”

“Most proteins that search DNA code can recognize one specific sequence merely by sensing the outside of the DNA double helix,” Elf continued. “Cas9 can search for an arbitrary code, but to determine whether it is in the right place, the molecule has to open the double DNA helix and compare the sequence with the programmed code. The incredible thing is that it can still search the entire genome without using any energy.”

The very high concentrations of Cas9 that are necessary for finding the right sequence within a reasonable time frame can pose severe problems for the cells that scientists try to alter. But since the nature of the search process is now understood, an important clue as to how the system can be improved has been found.

By sacrificing a portion of Cas9's flexibility, it would be possible to design genetic scissors that are still sufficiently versatile to edit various genes but simultaneously fast enough to be medically usable.

“The results have given us clues on how we might achieve that kind of solution,” noted Elf. “The key is in what are known as the 'PAM [protospacer adjacent motif] sequences,' which determine where and how often Cas9 opens up the DNA double helix. Molecular scissors that do not need to open the helix as many times to find their target are not only faster but would also reduce the risk of side effects.”







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