Modification doubles available sites of a process that knocks out genes
Release Date: August 8, 2014
By modifying an existing “genome editing” technology that allows precise modification of pieces of DNA from chromosomes, Johns Hopkins researchers report they have significantly increased the range of DNA sites that can be efficiently edited by the process. In a description of their novel advance, reported in the Aug. 8 issue of Nature Communications, they say the modified methodology could eventually add efficiency and speed studies of gene function, aid in the development of new cellular models of diseases, and help treat genetic conditions.
The researchers, led by Vinod Ranganathan, Ph.D., a postdoctoral fellow in the Wilmer Eye Institute in the Department of Ophthalmology at the Johns Hopkins University School of Medicine, expanded on a relatively new way of modifying the genome known as CRISPR gene editing. The name CRISPR is an acronym for DNA segments known as clustered regularly interspaced short palindromic repeat, which refers to the biological origin of the method.
CRISPR technology, which was first used as a gene editing tool in 2013, takes advantage of an immune function that bacteria use to battle viruses. The system relies on an enzyme, CAS9, that cuts DNA at a desired site after being directed to the appropriate site by short “guide” RNA molecules that recognize the desired DNA sequence.
CRISPR technology is able to achieve DNA editing in weeks or months, making it far more efficient and faster than previous methods that take months to years. However, current CRISPR methodology is limited by the guide RNAs that are used to direct the DNA cutting, according to Ranganathan. “Rather than being able to direct the enzyme to cut the genome at any site — and using these cuts to disable genes or as open sites to insert new ones — the RNA that researchers have typically used can only place the cutting enzyme at a limited subset of sites,” he notes.
Seeking a way to get access to more of the genome, Ranganathan and his colleagues modified the way in which guide RNAs are generated, making it possible to synthesize guide RNAs that begin at either of two of the nucleotides, adenine (A) or guanine (G), rather than just at G. “Since 15 percent 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,” Ranganathan says.
To test out their improved CRISPR methodology, 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 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,” says 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 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.”
Other Johns Hopkins researchers who participated in this study include Karl Wahlin and Julien Maruotti.
This work was supported by the National Institutes of Health’s National Eye Institute (5T32EY007143,R01EY009769 and 5P30EY001765), the Maryland Stem Cell Research Foundation, Foundation Fighting Blindness, Bright Focus Foundation, Research to Prevent Blindness, and generous gifts from the Guerrieri Family Foundation and from Mr. and Mrs. Robert and Clarice Smith.