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Insight & Intelligence™ CRISPR/Cas Comes of Age–Almost Patricia Fitzpatrick Dimond, Ph.D. In some prokaryotes, clustered regularly interspaced short palindromic repeats (CRISPR) and their CRISPR-associated (Cas) proteins make up a defense system against bacteriophages and plasmids. CRISPR DNA loci typically consist of several noncontiguous direct repeats separated by stretches of variable sequences, or spacers that correspond to captured viral and plasmid sequences, most often adjacent to Cas genes. Cas genes encode a family of proteins that carry functional domains typical of nucleases, helicases, polymerases, and polynucleotidebinding proteins. To date, investigators have discovered a lot about the function of Cas proteins by disabling Cas genes, thereby demonstrating they are required for immunity in addition to the CRISPR sequences. Disabling the Cas7 gene, for example, disrupts the cell's ability to incorporate new spacers; disabling the Cas1-like gene causes a loss of resistance against phages, even if the relevant spacers are present. Four Cas protein families, designated Cas1 to Cas4, are strictly associated with CRISPR elements and always occur near a repeat cluster. Computational biologists from The Institute for Genomic Research have recently systematically investigated uncharacterized proteins encoded in the vicinity of these CRISPRs, discovering many additional protein families that are strictly associated with CRISPR loci across multiple prokaryotic species. The scientists built multiple sequence alignments and hidden Markov models for 45 Cas protein families. Based on their studies, the investigators concluded thus far that CRISPR/Cas gene regions can be very large—with up to 20 different, tandem-arranged Cas genes next to a repeat cluster or flling the region between two repeat clusters. Distinctive subsets of the collection of Cas proteins recur in phylogenetically distant species and correlate with characteristic repeat periodicity. The investigators say their analyses support initial proposals of mobility of these units, along with the likelihood that loci of different subtypes interact with one another as well as with host cell-defensive, replicative, and regulatory systems and that CRISPR/Cas loci are larger, more complex, and more heterogeneous than previously thought. Genetic Engineering From a practical standpoint, the CRISPR system's ability to precisely and reliably cleave DNA has made it an active area of study for purposes of genetic engineering. Breakthroughs in understanding the mechanisms of the CRISPR/Cas, scientists say, offer great potential for biotechnological applications and understanding evolutionary dynamics. In particular, CRISPRs can be designed and customized to induce cuts at precise location in the genome. Unlike other tools, CRISPRs are constructed from RNA—a cheaper and easier starting material—and can make nicks simultaneously at more than one genomic location, allowing researchers to look at the effects of combinations of mutations. In 2012, University of Zürich's Martin Jinek, Ph.D.— who was then at the University of California, Berkeley—and his colleagues reported in Science research results showing that in a subset of CRISPR/Cas systems the mature CRISPR RNA (crRNA) that is base-paired to transactivating crRNA (tracrRNA) forms a two-RNA structure that directs the CRISPR-associated protein Cas9 to introduce double-stranded breaks in target DNA. At sites complementary to the crRNA-guide sequence, the Cas9 HNH nuclease domain cleaves the complementary strand, whereas the Cas9 RuvC-like domain cleaves the noncomplementary strand. The dual-tracrRNA:crRNA, when engineered as a single RNA chimera, also directs sequencespecifc Cas9 double-stranded DNA cleavage. The authors said their study revealed a family of endonucleases that use dual RNAs for site-specifc DNA cleavage and highlights the potential to exploit the system for RNAprogrammable genome editing. Last January, the Broad Institute's Feng Zhang, Ph.D., and colleagues at MIT and Rockefeller University reported having engineered two different type II CRISPR systems, and demonstrated that Cas9 nucleases could be directed by short RNAs to induce precise cleavage at endogenous genomic loci in human and mouse cells. Further they said, Cas9 could also be converted into a nicking enzyme to facilitate homologydirected repair with minimal mutagenic activity. Importantly, multiple guide sequences could be encoded into a single CRISPR array to enable simultaneous editing of several sites within the mammalian genome, demonstrating easy programmability and wide applicability of the CRISPR technology. "All of the mutations that people identify through genomewide association studies are based on clusters of mutations. You can't test that by introducing just one mutation at a time into a cell," says Dr. Zhang. "You have to test all of them together because they may be interacting with each other." Dr. Zhang and his colleagues are pursuing ways to create 140 Huguenot Street, New Rochelle, NY 10801-5215 914 740-2100 www.GENengnews.com Publisher & CEO Mary Ann Liebert Editor in Chief John Sterling editor@GENengnews.com GEN Group Publisher Sande Giaccone MANAGING EDITOR PRODUCTION EDITOR SENIOR EDITOR TECHNICAL EDITOR ONLINE EDITOR SENIOR NEWS EDITOR ASSISTANT EDITOR ART DIRECTOR DIRECTOR, DIGITAL MEDIA AUDIENCE DEVELOPMENT ONLINE PRODUCT MANAGER WEB PRODUCER SALES ADMINISTRATOR OFFICE ASSISTANT Tamlyn L. Oliver Robert M. Reis Kevin Mayer Patricia F. 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