Small Molecules to enhance HDR
I did a literature search tailored to find ways to increase HDR in Human IPS - ES cells. I was interested in trying drugs to enhance Homology directed repair.
Has anyone has tried any of the following drugs- constructs in their lab
or know of any which I have left off the list.
I will be introducing a SNP into an IPSC line and historically in my lab the efficiency has been very bad .1% realistically. However we don't electroporate, we are using Lipofectamine and RNP complexes from IDT. Before that we were using Plasmid vectors. IPSCs are giving us a lot of trouble to the point that some of us are resorting to transfecting fibroblasts with hopes to reprogram later into their IPSC disease model. Let me know if you have tried any of the following drugs or techniques, or if your lab uses a drug or technique off the list.
I have tried all the chemicals including the ones you did not include here.
My results are all based on human cancer cell lines, not iPSCs or ESCs.
Therefore, please be advised that it could be different in your cells.
SCR7 never worked in any types of cells tested.
Nocodazole gave me 2~3 fold increase in HR in multiple cell types.
L755507 never worked.
RS-1 did not work in a couple of cell lines tested.
DNA-PK inhibitors worked the best. Between NU and KU, I got better efficiency using NU. (several cell lines have been tested)
You can also use a little more NU compound compared the amount you can find in literature. I use 3X without seeing any toxicity.
Please remember that you need to get enough NHEJ just by doing CRISPR itself.
For example, if you get 10% indels without doing HR, you will get less than 10% of HR at the end since you are trying to get HR by antagonizing NHEJ.
I typically get 80-90% genome modification efficiency (measured by TIDE or Mi- seq) in NHEJ, then I get about 50% HR efficiency by using NU compound.
I also strongly recommend electroporation method over lipofectamine.
I get 98% genome editing in HEK293 in most cases and >70% in other cells including primary cells using electroporation.