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Pair of Studies Report Development of Cas9-based Genome Editing Tools

NEW YORK (GenomeWeb News) – A pair of studies that appeared online today in Science demonstrated that repeats found in bacterial genomes that act as a sort of bacterial immune system can be repurposed as a tool to enable genome editing.

The two sets of researchers independently drew upon the ability of the clustered regularly interspaced short palindromic repeats, or CRISPR, system to direct, site-specific cleavage of DNA using short RNAs. In particular, both groups turned to the CRISPR-associated CAS9 nuclease to form the basis of their genome editing tools.

Such tools, they said, could be applied not only to basic research but potentially to medicine.

"We expect RNA-guided genome targeting to have broad implications for synthetic biology, the direct and multiplexed perturbation of gene networks, and targeted ex vivo and in vivo gene therapy," wrote Harvard Medical School's Prashant Mali and his colleagues in their paper.

Mali and his team engineered a human-codon-friendly version of Cas9 with an SV40 nucleus-localization signal tacked on to its C-terminus. Additionally, they developed a CRISPR RNA fused to a trans-activating CRISPR RNA, together termed guide RNA, or gRNA, to direct Cas9 to cleave specific sequences.

They then tested their creation in a cell line containing a GFP reporter construct that would light up when the AAVS1 locus their gRNAs, called T1 and T2, were targeting underwent homologous recombination. Using flow activated cell sorting, they noted gene correction rates near 3 percent and 8 percent for their two gRNAs targeting the AAVS1 locus. They added that the editing process using Cas9 appeared to be faster than an alternative genome editing approach based on TAL effector nucleases, or TALENs.

The researchers then tested their tool in cell lines that usually express AAVS1, and there, too, they found non-homologous end-joining rates that ranged between 2 percent and 25 percent, depending on the cell line.

Further, Mali and his colleagues were able to then use their approach to integrate both a double-stranded DNA donor and an oligo DNA donor into the AAVS1 locus. "These results demonstrate that Cas9 is capable of efficiently integrating foreign DNA at endogenous loci in human cells," they wrote.

The Harvard group also developed a resource of about 190,000 sequences in the human genome that can be targeted by gRNA, and it incorporated those sequences onto DNA arrays for others to be able to use.

Meanwhile, Le Cong from the Broad Institute and the Massachusetts Institute of Technology and his colleagues engineered a similar tool, also based on the Cas9 nuclease. To do this, they also attached a nuclear-localization signal to Cas9 and to RNaseIII. Additionally, they expressed a tracrRNA under the direction of the RNA polymerase III U6 promoter, and, also under the direction of a U6 promoter, expressed a pre-crRNA array with a single guide space flanked by direct repeats. The original guide targeted the human EMX1 locus.

They then tested different components of the CRISPR system in a cell line, finding that three aspects of the system — Cas9, tracrRNA, and pre-crRNA — were needed for cleavage.

They further tested their system in a eukaryotic system, targeting the EMX1 locus, and then examined a crRNA:tracrRNA hybrid design targeting the human PVALB locus and mouse Th loci in human and mouse cells. "We achieved efficient modification at all three mouse Th and one PVALB targets using the crRNA:tracrRNA design, thus demonstrating the broad applicability of the CRISPR system in modifying different loci across multiple organisms," they wrote.

"That's the beauty of this — you can easily program a nuclease to target one or more positions in the genome," senior author Feng Zhang added in a statement.

The Broad group said it will be making its reagents and software tools available through Addgene, and they noted that they have filed a patent application based on this work.

While the researchers say that Cas9-based tools for genome editing are more precise than TALEN or zinc-finger based approaches, the tools are constrained by the need to have a protospacer-adjacent motif near the target and by the specter of nuclease off-target effects.

"Elucidating the frequency and underlying causes of off-target nuclease activity induced by CRISPR, ZFN, and TALEN genome engineering tools will be of utmost importance for safe genome modification and perhaps gene therapy," Mali et al. wrote. "Potential avenues for improving CRISPR specificity include evaluating Cas9 homologs identified through bioinformatics and directed evolution of these nucleases toward higher specificity."

If it works, though, the researchers said such genome editing could have widespread applications. "The ability to carry out multiplex genome editing in mammalian cells enables powerful applications across basic science, biotechnology, and medicine," Cong and his colleagues wrote.

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