Takayama et al (2017) describe in Nucleic Acids Research how they increased the frequency of biallelic editing of genes in human ES/iPS cells and their findings may be useful to insect biologists.
The efficiency of Cas9-mediated homologous recombination (HR) can vary quite a bit and some of the factors important in determining this efficiency are known. For example, at least in human embryonic stem cells and induced pluripotent stem cells, there is a correlation between efficiency of HR at a locus and the expression levels of the gene, with transcriptionally active genes more efficiently targeted than transcriptionally inactive genes.
Takayama et al. were interested in finding ways to enhance HR in human embryonic stem cells to increase the frequency of biallelic Cas9-mediated gene editing in general and of transcriptionally inactive genes in particular. Others have also been interested in finding such enhancements and these have included reducing non-homologous end joining by silencing ligase IV and or Ku80 either by mutation or RNAi. The use of small molecules such as the Ligase IV inhibitor Scr7 can also be effective.
Takayama et al. initally targeted two genes – one actively transcribed and the other not transcribed. The authors inferred that the transcriptionally inactive gene was in chromatin with numerous epigenetic marks associated with closed chromatin. The authors screened a small-molecule library and found that valporic acid (VPA) could increase the efficiency of HR if cells were pretreated with it before being transfected with a cocktail of CRISPR/Cas9 plasmids and donor plasmids. The effect was approximately a 2 fold increase of HR at both loci.
It has been known that overexpression of RAD51, a molecule that plays an important role in HR of DNA during double strand break repair, increases HR efficiency 2-3 fold in CHO cells. Takayama et al. found that overexpressing RAD51 also increased the efficiency of CRISPR/Cas9-induced biallelic gene editing.
Interestingly the authors report synergistic effects of VPA and overexpression of RAD51 and confirmed this effect with editing experiments at 11 other loci.
How well these finding will transfer to insects remains to be seen but it appears the options for enhancing HR during editing experiments has increased.
Kazuo Takayama, Keisuke Igai, Yasuko Hagihara, Rina Hashimoto, Morifumi Hanawa, Tetsushi Sakuma, Masashi Tachibana, Fuminori Sakurai, Takashi Yamamoto, Hiroyuki Mizuguchi; Highly efficient biallelic genome editing of human ES/iPS cells using a CRISPR/Cas9 or TALEN system. Nucleic Acids Res 2017; 45 (9): 5198-5207. doi: 10.1093/nar/gkx130