Two recent papers, Maruyama et al (2015) and Chu et al. (2015), describe the use of a small molecule inhibitor of non-homologous end-joining known as Scr7 to increase rates of homology directed repair (HDR) triggered by double stranded breaks in DNA by Cas9.
Scr7 could be useful to insect scientists working with CRISPR/Cas9 technologies.
CRISPR/Cas9 mediated genome manipulations now include a wide array of options beyond ‘simple’ knock-outs or knock-ins.
Nonetheless, knock-outs and knock-ins are the most common applications and because knock-ins rely on HDR they are less frequent than knock-out mutations that rely only on non-homologous end-joining (NHEJ).
Various approaches have been reported to inhibit or suppress NHEJ to increase rates of HDR including silencing genes encoding proteins involved in NHEJ like Ligase IV, Ku80 and Ku70 with dsRNA or by working in genetic backgrounds deficient in NHEJ (Ligase IV mutants).
Maruyama et al (2015) work with human cells in culture and with mouse embryos. In both systems they show significant increases in HDR in the presence of Scr7, up to 19-fold improvement in one case. Maruyama et al. acknowledge the possible widespread applicability of this approach, depending on the degree of conservation of NHEJ machinery (Ligase IV) in your system of interest.
Particularly interesting is Maruyama et al’s inclusion of Scr7 in an injection mix they used to inject mouse zygotes, which is very much analogous to how insect scientists might apply this compound in a knock-in experiment.
Chu et al. (2015) perform all of their studies in human cell culture using fluorescence activated cell sorting (FACS) analysis to measure outcomes. They look not only at Scr7 but also at two proteins from adenoviruses that are known to mediate ubiquintination and proteasomal degradation of Ligase IV.
Chu et al. (2015) find that all of their approaches to reducing Ligase IV activity in cells increased HDR.
Scr7 is a small molecule/anti cancer drug that interacts with Ligase IV and interferes with its DNA binding but not its ligase activity. See this reference for some of the initial description of the biological activity of Scr7, including its anti-cancer activity.
Scr7 is widely available commercially at affordable prices and should it prove effective in insect systems it will be an easy way to increase rates of HDR. Currently, Ligase IV mutants have been used as hosts for HDR experiments (Drosophila melanogaster) or dsRNA to silence Ligase IV gene expression has been included in injection cocktails. Addition of dsRNA tends to increase the viscosity of injection cocktails, making injections somewhat more challenging so eliminating the need for dsRNA would be advantageous. Avoiding the need to create and maintain ligase IV knock-out mutants could also be an advantage in using Scr7.
Ligase IV is fairly well-conserved so there is a chance Scr7 will be effective against Ligase IV from various insects.
Time will tell. These papers are worth a look if you are using CRISPR/Cas9 in insects.
Takeshi Maruyama,Stephanie K Dougan,Matthias C Truttmann,Angelina M Bilate,Jessica R Ingram& Hidde L Ploegh (2015) Increasing the efficiency of precise genome editing with CRISPR-Cas9 by inhibition of nonhomologous end joining. Nature Biotechnology, Published online. 23 March 2015 doi:10.1038/nbt.3190
Van Trung Chu, Timm Weber, Benedikt Wefers, Wolfgang Wurst, Sandrine Sander, Klaus Rajewsky & Ralf Kühn (2015) Increasing the efficiency of homology-directed repair for CRISPR-Cas9-induced precise gene editing in mammalian cells. Nature Biotechnology, Published online 24 March 2015 doi:10.1038/nbt.3198