September 1, 2013 (Vol. 33, No. 15)

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 polynucleotide-binding 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 filling 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 sequence-specific Cas9 double-stranded DNA cleavage.

The authors said their study revealed a family of endonucleases that use dual RNAs for site-specific DNA cleavage and highlights the potential to exploit the system for RNA-programmable 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 homology-directed 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 genome-wide 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 and test many precise mutations at once using CRISPR.

New Applications, Unintended Effects

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 significant 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 Cas9-based 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 five 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 RNA-DNA interfaces in human cells, a finding 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 significant 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 specificity 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.

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