I am aware of the triple mutated version (K219A-D362A-D384A) of PINK1, however, I am looking to do a CRISPR into human neuronal cells and the simpler, the better the approach. I have seen that the E240K abolishes the TcPINK1 kinase activity but nothing in human, not even OE. If anyone can share something useful it would be greatly appreciated
I don't know the answer to this specific gene but here is how I would approach it. First, if the active site(s) is/are known, you would want to try to introduce a mutation there. Typically, a mutation in a phosphorylated amino acid can be used. Sometimes, in natural mutations, you can introduce a bulky amino acid that will block the active site. The bcr/abl translocation for CML has been widely studied and drugs and mutations that inactivate it.
If no active site is known, I would do a homology study for both human and non-human enzymes and look for known active sites or those homologous to active sites.
A way that is not often used is to have someone do mutational studies in yeast or Drosophila.
Of course you need a way to assay the enzymatic activity.
This may be too general to help you, but thought I would mention in case it helps you or someone else.
Hi Bernardo
for human you could go to the UCSC website and explore its genome browser with some optional tracks as OMIM to get more actions on your field:
try that: https://genome-euro.ucsc.edu/cgi-bin/hgTracks?db=hg19&lastVirtModeType=default&lastVirtModeExtraState=&virtModeType=default&virtMode=0&nonVirtPosition=&position=chr1%3A20955433%2D20982518&hgsid=320131203_yBQZYwLis3XiKFv0iS6PPwa351zU
all the best
fred
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