Hi,

does anybody produce CRISPR RNA-guided endonuclease Cas9 from S.pyogenes?

We would like to produce it with an N-terminal NLS of SV40 or similar. According to Methods in Enzymology, Volume 546 (2014) from Carolin Anders & Martin Jinek, they poduce it in Rosetta 2 DE3 with a N-terminal His6-MBP fusion tag and cleave with TEV protease.

Any suggestion?

Hi Mario,

Our mutant mouse facility got a clone from Addgene that is the S.pyogenes pET-NLS-Cas9-6xHis construct. It expresses very well in E.coli and has similar (or better) in vitro activity to that obtained from commercal sources. It is soluble so I am not sure that an MBP fusion is necessary, and will just give you more work,  and the expression and purification protocols are also (I think) linked on the Addgene site.  Peggy is also producing it so if you need anything else you have (at least) two P4EU labs working with it-I am sure there are others.

Testing in cells is under way but we don't have that data yet.

Hi Mario,

If I am well informed Anja Schuetz in Berlin also has a clone to produce Cas9 in high amounts. You can contact her.

Good luck

Joop