Researchers at the Broad Institute of MIT and Harvard and at the McGovern Institute for Brain Research at MIT have engineered changes to the revolutionary CRISPR/Cas9 genome editing system that significantly cut down on "off-target" editing errors. The refined technique addresses one of the major technical issues in the use of genome editing. The CRISPR/Cas9 system works by making a precisely targeted modification in a cell's DNA. The protein Cas9 alters the DNA at a location that is specified by a short RNA strand whose sequence matches that of the target site. While Cas9 is known to be highly efficient at cutting its target site, a major drawback of the system has been that, once inside a cell, Cas9 can bind to and cut at additional sites that are not targeted. This has the potential to produce undesired edits that can alter gene expression or knock a gene out entirely, which might lead to the development of cancer or other problems. In a paper published online on December 1, 2015 in Science, Feng Zhang, Ph.D., and his colleagues report that changing just 3 of the approximately 1,400 amino acids that make up the Cas9 enzyme from S. pyogenes dramatically reduced "off-target editing" to undetectable levels in the specific cases examined. The Science article is titled “Rationally Engineered Cas9 Nucleases with Improved Specificity.” Dr. Zhang and his colleagues used knowledge about the structure of the Cas9 protein to decrease off-target cutting. DNA, which is negatively charged, binds to a groove in the Cas9 protein that is positively charged. Knowing the structure, the scientists were able to predict that replacing some of the positively charged amino acids with neutral ones would decrease the binding of "off target" sequences much more than the binding of "on target" sequences.
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