Hello.

I'm planning to perform the plasmid transformation efficiency using Cas9 protein in bacterial strain which has Type I-F and I-E CRISPR-Cas system.

I transformed the pCas9 coding the guide RNA into the strain and the backbone plasmid, target plasmid were transformed into the that strain.

I predict that the transformation efficiency of the backbone plasmid group is higher than the target group but, the reverse result was shown, almost ten-thousands higher than backbone group.

Efficiency

Backbone : 100

Target : 20,000

I confirm the guide RNA sequence in pCas9 and the sequence was correct. The sequence of target plasmid (pBAD18::Target) was also confirmed and correct.

I suppose the failure reason of this experiment

1. the stability of Cas9?

Cas9 protein may be unstable in Type I CRISPR-Cas system

or the strain affects the pCas9 stability.

2. Target plasmid is improper in this experiment?

I used the pBAD18 (the compatibility was confirmed), but this vector is used for protein expression. So i am wondering that whether this plasmid is proper or improper in this experiment.

I consider that the stability of Cas9 in the strain is an important point.

I hope to get any advice or discussion. Thank you.