Dear Ayesha, CRISPR proteins are very sensitive. First thing you should check is codon optimization for e coli. Second is expression at low temperatures with induction at higher OD600 (around 0.8), usually low concentrations of IPTG work better (mM range). Using protease inhibitors, sonication, and sometimes TB media can also help.

Good luck.

thank you for your reply... i added iptg in low concentration ( ranging from 0.1mM to 1 mM )and also used protease inhibitors... but no protein is produced. i will try with TB media... Thank you so much for your consideration

Hello Ayesha, it should be work by follow-

1. Use temperature range from 16'C, 22'C and 37

2. Try to use 0.5-0.8 OD600 grown secondary culture from induction also use above 1 OD because some proteins show inducuction at higher OD like 1 or more.

3. Use freshly prepared IPTG

4. Codon Optimization

5. Try in different cells like Roestta, BL21(DE3),BL21(Lys) etc.

Thank you for your time and consideration