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CRIPSR-Cas9 modification improves genome editing precision
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The CRISPR-Cas9 nucleases are increasingly commonly used genome editing tools with potential for future therapeutic application. However, the monomeric versions up to use until now are prone to processing errors in which they produce unwanted off-target mutations at a significant rate, making it inappropriate for use in a clinical setting. A new study from researchers in Massachusetts General Hospital describes an important modification of this system what substantially improves the precision. The paper is published online in the journal Nature Biotechnology.

The CRISPR-Cas9 nucleases combine a short RNA segment that matches a relevant DNA target with Cas9, a DNA-cutting enzyme. They were initially developed in 2012 and are easier to use than earlier genome editing systems such as ZFN (zinc finger nuclease) and TALEN (transcription activator-like effector nuclease) systems. They have been used in animal models and human cells to induce genomic changes. However, previous reports from the laboratory where the current study was carried out had shown that CRISPR-Cas nucleases produced additional mutations in human cells at high frequency, even at sites differing from the intended DNA target by as many as five nucleotides.

In the current study, the targeting activity of Cas9 was fused to a nuclease called FokI. FokI only cuts when two guide RNAs are bound to the DNA within a strictly defined distance and orientation, a process known as dimerization. Thus the new nucleases were termed dimeric RNA-guided FokI nucleases (RFNs). The need for dimerization effectively doubled the length of DNA that must be recognized for cleavage by RFNs. This significantly improved precision of genome editing without any sacrifice of on-target modification effectiveness.

Senior author Dr J. Keith Joung explained the importance of this modification for future therapeutic applications that require high precision in genome editing: "This system combines the ease of use of the widely adopted CRISPR/Cas system with a dimerization-dependent nuclease activity that confers higher specificity of action…..Higher specificity will be essential for any future clinical use of these nucleases, and the new class of proteins we describe could provide an important option for therapeutic genome editing." The researchers have also developed a software application that will help users identify potential RFN target sites. They have incorporated it into the freely available software package ZiFiT Targeter, which can be accessed at http://zifit.partners.org.

Sources:

Tsai, S.Q., Wyvekens, N., Khayter, C., Foden, J.A., Thapar, V., Reyon, D., Goodwin, M.J., Aryee, M.J. and Joung, J.K. Dimeric CRISPR RNA-guided FokI nucleases for highly specific genome editing. Nature Biotechnology (2014); doi:10.1038/nbt.2908

Press release: Massachusetts General Hospital; available at http://www.eurekalert.org/pub_releases/2...042514.php
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