Hi I want to introduce a missense mutation via CRISPR-Cas9 via HDR. I will transfect my cells with a guide-RNA and Cas9 (encoded by one plasmid) and want to cotransfect a ssODN (1 ug). I have checked the guide RNA in HEK293 cells and it is working (T7 assay). Then I want to do FACS and want to sort cells in 96well plates (one cell per well).
To predict how many cell colonies I have to screen, it would be helpful to know how many colonies will presumably repair the double strand break via NHEJ or over homology-directed repair (HDR)?
Does anyone has tipps how I can improve the ratio for HDR? Thanks for answering! Andreas
Hello Andreas,
the success rate depends on many factors, but it should be above 1% for HDR usually. Therefore, one 96 well plate should work for the first try. Some companies offer to purify and sequence whole 96 well plates (to save you work).
There are also various concepts for increased HDR efficiency. You could try to add the ligase IV inhibitor SCR7, which will suppress NHEJ (doi:10.1038/nbt.3190 , doi:10.1038/nbt.3198).
Good luck!
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