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 이성욱 ( 2014-08-09 00:59:43 , Hit : 2140
 New Guide RNAs for CRISPR Can Double the Targetable Genome

GEN News HighlightsMore »
Aug 8, 2014

Kevin Mayer New Guide RNAs for CRISPR Can Double the Targetable Genome Source:
© Kirsty Pargeter/Fotolia.com

(Page 1 of 1)
Since it burst upon the gene editing scene, CRISPR technology has dazzled researchers with its possibilities. Yet some researchers are so dissatisfied with CRISPR’s limitations that they are doing their utmost to enhance CRISPR’s already prodigious powers.
For example, researchers at Johns Hopkins realized that current CRISPR methodology is limited by the guide RNAs that are used to direct the DNA cutting. With CRISPR, a guide RNA works with an enzyme, Cas9, to cut DNA at a particular site. This approach is powerful because guide RNAs can be constituted to recognize different DNA sequences.
The problem is, given the current technology for generating guide RNAs, gene editing scientists must be content with synthesizing guide RNAs that begin at the nucleotide guanine (G). But now, say the Johns Hopkins researchers, it may be possible to generate guide RNAs that start not only at G, but at adenine (A).
“Since 15% more genes that have target sites start with an A rather than a G, simply using this expanded guide RNA approach more than doubles the number of sites we can access,” said Vinod Ranganathan, Ph.D., a Johns Hopkins postdoctoral fellow who led the research.
Dr. Ranganathan is also the first author of a study that appeared August 8 in Nature Communications. This study—“Expansion of the CRISPR-Cas9 genome targeting space through the use of H1 promoter-expressed guide RNAs”—describes how the Johns Hopkins team modified the CRISPR gene editing technique.
“gRNA expression through the commonly used U6 promoter requires a guanosine nucleotide to initiate transcription, thus constraining genomic targeting sites to GN19NGG,” the authors wrote. “We demonstrate the ability to modify endogenous genes using H1 promoter-expressed gRNAs, which can be used to target both AN19NGG and GN19NGG genomic sites.”
To test their improved CRISPR methodology, Dr. Ranganathan and his colleagues used it to target a gene inserted in various cell lines that made the cells glow green. They found that the new CRISPR method effectively cut the gene, disabling it just as well as the old CRISPR method.
The team expanded on this experiment by using CRISPRs to mutate a gene responsible for causing a form of a blinding eye disease known as retinitis pigmentosa in human stem cells. Sequencing the genes from these cells afterward showed that their technique was successful, suggesting its utility for studying or eventually treating this and any number of other genetic disorders.
The researchers’ further analysis showed that target sites in the genome beginning with A are more often found near disease genes, making the modified CRISPR even more useful for targeting areas of DNA that researchers find most interesting.
“This new method gives us a lot more flexibility for genetic engineering,” said research team leader Donald Zack, M.D., Ph.D., the Guerrieri Family Professor of Ophthalmology in the Center for Genetic Engineering and Molecular Ophthalmology at the Wilmer Institute and Dr. Ranganathan’s mentor. “The old technique is like an express train that can only make stops every few miles along DNA. This new technique is like a local train—we can generate mutations more efficiently than we ever could before.”
Concluding remarks from the Nature Communications paper take stock of CRISPR (an acronym for DNA segments known as clustered regularly interspaced short palindromic repeat, which refers to the biological origin of the method), emphasizing its demonstrated utility and anticipating where it may yet go.
“With enhanced CRISPR targeting through judicious site selection, improved Cas9 variants, optimized gRNA architecture, or additional cofactors, an increase in specificity throughout the targeting sequence will likely result, placing greater importance on the identity of the 5′ nucleotide,” the authors predicted. “As a research tool, this will allow for greater manipulation of the genome while minimizing confounding mutations, and for future clinical applications, high-targeting densities coupled with high-fidelity target recognition will be paramount to delivering safe and effective therapeutics.”







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