Hi friends,
I am trying to knock-out a goi but whenever I try to design cas9 gRNA it is turning to a disappointing event. I have full sequence of my goi and KEGG map data for metabolism of enzyme pathway. But my organism is not a model one in design tools. Also the end product is unique for that species. So I couldn’t manage to design by using any online tools so far. Do you have any suggestions for it?
Thanks,
Dr. Deniz KOM
Dear Deniz,
First of all try to search out for appropriate online tools that have your model organism into database (There are many tools available, some are free and some are paid). Secondly, if your model organism is not present in any database then try to look for same homologous GOI in nearest related organism present in database. Importantly in second you must have to be sure that your GOI is more than 95% identical to the nearest organism. If even this doesn't works out for you then manually make the list of all possible gRNAs for your GOI from both sense and anti-sense strand and once you have all of them check for the off target site efficiency of each any every individual gRNA in the genome of your organism (there are several tools for this purpose as NCBI etc).
It is always better to choose gRNA having 0 off target site or as minimum as possible. If there are no gRNA with 0 off target site then choose the gRNAs having mismatch nucleotides in a close vicinity of PAM site.
Hope this works out for you
Best Wishes
Please, for advices visit:
https://www.researchgate.net/post/How_easy_difficult_is_the_use_CRISPR_Cas9_system_for_functional_genomics_in_non-model_insects
=)
- Badges
- Science topic
- Similar topics
- Biological Science
- Biochemistry
- Enzymology
- Enzymes