They confirm what Singh et al previously reported in a small but exciting data morsel last fall, which is that substantially higher rates of HDR-mediated insertion can be achieved in mouse zygotes by treating them with the NHEJ inhibitor, SCR7, during the injection process. They actually mixed SCR7 (final conc. 1mM) directly into the injection cocktail of gRNA + Cas9mRNA + donor ssDNA oligo. After some preliminary tests in cell lines, they moved to zygotes. Using a donor oligo to insert a short peptide tag and validated CRISPR targets/gRNAs, they did tests with and without SCR7. Bottom line: HDR-mediated insertion rates increased by several fold for the two genes they tested. Although that may not sound like a breakthrough to some of you, many of you will know that in the world of mouse engineering it's key, because it will probably often mean the difference between getting zero versus a few correctly engineered pups out of an injection series. Some other highlights are: 1. The embryos seem to tolerate SCR7 application under these conditions with no problem; no toxicity or increased death was noted. Various other studies seem to support that transient inhibition of NHEJ is well tolerated. Note that the SCR7 target, ligase IV, is critical for embryonic development so it can't be globally knocked out. 2. No increase in off-target effects. Cool. Technical notes: 1. Yesterday's google searching quickly turned up 3 companies selling SCR7. Yay. 2. SCR7 must be dissolved in DMSO. I think making a stock solution of 100 mM SCR7 in DMSO is reasonable. So the final injection mix, with 100-fold SCR7 dilution from the stock, will have 1 mM DMSO and also 1% DMSO. I couldn't dig out the SCR7 stock solution details from the paper but it's probably close to these parameters. 3. SCR7 very strongly inhibits the recovery of NHEJ-style mutations from the CRISPR targets. 4. The zygote injections were all done cytoplasmic, not pronuclear, although they were done at the pronculear stage. Thus it is clear that HDR edits with ssDNA oligos can be efficiently done by cytoplasmic injections. This is great because it results in higher rates of pup survival than pronuclear injection. Still lingering questions for me: 1. Although the authors showed they could increase the insertion rate of a "large" cassette - a GFP-style reporter ORF - in cell culture, they did not repeat this experiment in embryos. Or at least they didn't show the data. Was there negative data to report? Or just not enough live pups yet for them to feel comfortable with publishing a negative result? Or have they not tried it yet? The routine insertion of kilobase-sized cassettes in embryos is now my next CRISPR mountain to climb! 2. I would kinda like to know if there may be an increased rate, or change, in the genome-wide mutation rate by SCR7 treatment. After all we are mucking around with the DNA repair pathway here. Since each mammal embryo probably has on the order of 50-100 new mutations anyway, it would have to be a pretty substantial change in mutation rate to scare me off. I'll bet there is no detectable effect. Besides, NHEJ usually results in new mutations anyway - so I would imagine that we'd observe cell or embryo death following SCR7 treatment, long before we could observe a change in mutation rates or spectrum in surviving embryos.
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.
Far above Cayuga's waters - my alma mater! - there are some excellent scientists using CRISPR to engineer mutations in mice. This paper is from John Schimenti's lab at Cornell and I believe it contains the most thorough review of mouse CRISPR engineering to date.
More than merely a review article, it has some additional new data from this group. They tested whether inhibition of the NHEJ pathway could enhance efficiency of CRISPR-mediated homology-directed repair, since these pathways compete following CRISPR cleavage; this was based on similar experiments done in Drosophila using ZFNs to do HDR. The answer seems to be yes, it helps in mouse embryos as well. The compound they used was SCR7, an inhibitor of ligase IV (a key player in NHEJ). Crucially, it seems that SCR7 can be applied directly to mouse embryo culture media with minimaltoxicity. Go to Table 2 for the result suggesting a shift in balance from predominantly NHEJ alleles toward more HDR alleles.
