New #CRISPR paper with more evidence that inhibiting NHEJ increases homology-mediated DNA repair. Improvements over SCR7.

Chu et al. have recently reported in Nature Biotechnology a detailed description of inhibiting the NHEJ pathway to enhance HDR (homology-directed repair) in cell culture CRISPR editing experiments.  This follows on the observation by Singh et al that SCR7, an inhibitor of ligase IV, accomplishes this in mouse embryos although the data set in the Singh paper was limited.  This new paper is very detailed in its implementation of a traffic-light reporter system to quantify NHEJ and HDR under various conditions.   

Increasing the efficiency of homology-directed repair for CRISPR-Cas9-induced precise gene editing in mammalian cells.  Van Trung Chu, Timm Weber, Benedikt Wefers,Wolfgang Wurst, Sandrine Sander, Klaus Rajewsky & Ralf Kühn. NatBiotechnol. 2015 Mar 24. doi: 10.1038/nbt.3198. [Epub ahead of print]

Since there's more than one way to potentially inhibit NHEJ, the authors used several approaches.  First, shRNA was used to silence ligase IV and/or KU70 or KU80.    The latter two proteins make up the KU heterodimer, required for NHEJ.     Second, they used SCR7.  Third, they coexpressed the adenovirus proteins E1B55K and E4orf6.   These are known target ligase IV for degradation. 

Before getting into more details, the bottom line was that each of these approaches (in human HEK293 cells) could both inhibit NHEJ and increase the rate of HDR at the same time.  However the approaches did this to varying degrees.  There was a clear trend that the more NHEJ inhibition occurred, the greater was the increase in HDR efficiency.   The most effective treatment was the coexpression of E1B55K + E4orf6.   Not only did this reduce NHEJ more than the other methods but it showed the greatest HDR increase as well.  

Figs. 1c, 2h, and 2j respectively suggest ~7x, ~8x, and ~3.4-51x increases in HDR were observed with E1B55K + E4orf6 coexpression as compared to the identical CRISPR/Cas experiments without these factors.

Therefore, transient NHEJ inhibition is looking better and better as a way to enhance HDR in CRISPR applications.  This will be likely be very important for gene therapy approaches, as well as just plain very useful for any HDR applications including of course, mouse embryo injections.   In fact, Chu et al go on to show that by combining flow-sorting of transfected cells with transient selection with an inserted antibiotic gene they were able to obtain essentially HDR-edited cell clones at essentially a 100% rate (e.g. supplemental fig. 13).  

Suddenly I am very interested in these adenoviral E1B55K and E4orf6 genes… These are apparently derived from the adenovirus C, a.ka. adenovirus 5, and Origene sells cDNAs and antibodies for these.    E4orf6 is also called E4orf6/7.