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Insight & Intelligence™ New Applications, Unintended Efects Apparently, the CRISPR/Cas system can also slow the spread of antibiotic resistance genes. Pathogenic bacterial strains emerge mostly due to transfer of virulence and antimicrobial resistance genes between bacteria through horizontal gene transfer. To study the impact of CRISPR on the emergence of virulence, Wenyan Jiang and colleagues at the Rockefeller University used the Cas9 endonuclease complexed with dual RNAs to introduce precise mutations in the genomes of Streptococcus pneumoniae and Escherichia coli. They programmed the human pathogen S. pneumoniae with CRISPR sequences that target capsule genes, an essential pneumococcal virulence factor, and showed that CRISPR interference can prevent transformation of nonencapsulated, avirulent pneumococci into capsulated, virulent strains during infection in mice. They further showed that bacteria can lose CRISPR function, acquire capsule genes, and mount a successful infection. The authors concluded that their results demonstrated that CRISPR interference can prevent the emergence of virulence in vivo and that strong selective pressure for virulence or antibiotic resistance can lead to CRISPR loss in bacterial pathogens. However, according to a commentary on Allele Blog, the enthusiasm about CRISPR/Cas was "somewhat dampened" by a June study in Nature Biotechnology that reported off-target effects of CRISPR/Cas was much higher than ZFN and TALEN. Researchers at Massachusetts General Hospital (MGH) reported that they had found a signifcant limitation to the use of CRISPR/Cas RNA-guided nucleases (RGN), the production of unwanted DNA mutations at sites other than the desired target. Yanfang Fu, Ph.D., and colleagues at MGH used a human cell-based reporter assay to characterize off-target cleavage of Cas9based RGNs. They found that single and double mismatches were tolerated to varying degrees depending on their position along the guide RNA (gRNA)-DNA interface. They also reported detection of off-target alterations induced by four out of six RGNs targeted to endogenous loci in human cells by examination of partially mismatched sites. The sites harbored up to fve mismatches and many were mutagenized with frequencies comparable to—or higher than—those observed at the intended on-target site. The investigators concluded that their work demonstrates that RGNs can be highly active even with imperfectly matched RNADNA interfaces in human cells, a fnding that might confound their use in research and therapeutic applications. "Recent work from our group and others has clearly demonstrated that CRISPR RNA-guided nucleases can have signifcant Comment on this article at www.GENengnews.com off-target effects in human cells. However, I don't view this as a limitation but rather just a parameter of the existing system that potential users need to account for in their experiments," J. Keith Joung, M.D., Ph.D., associate chief for research in MGH's department of pathology and co-senior author of the report, told GEN. "For example," he continued, "researchers who use these tools should perform appropriate controls to ensure that any effects or phenotypes that they observe are due to the on-target gene sequence or expression change and not to an off-target effect." Dr. Joung added that CRISPRs still show promise. "Currently, it is clear that the system makes a powerful research tool for altering gene sequence or gene expression. It will be interesting to begin to explore the use of these reagents for therapy, carefully taking into account the potential for off-target effects as these applications are explored," he said. He and others are working to improve the specifcity of CRISPR-based reagents with the hope that advances in the platform will also maintain its current ease-of-use. Another equally important effort of his group, Dr. Joung said, is directed at the development of methods that will enable us to assess the genome-wide effects of CRISPR reagents in an unbiased fashion. improved binding capacity To help increase throughput and precision in immunoassay and molecular biology applications, Thermo Scientific Sera-Mag SpeedBeads Magnetic Particles feature a second layer of magnetite to respond twice as fast in a magnetic feld as compared to our original Sera-Mag particles. In addition to low, non-specifc binding, these nominal 1 μm particles feature a unique, caulifower-like surface that provides a much larger area for binding reactions than smooth surface particles. quick magnetic response time • drive discovery at www.thermoscientific.com/particletechnology New! 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