I am looking for a bacterial expression vector for bacterial genome editing but thank you for your info.

I think, they provide the second plasmid only for doing targeting in parallel, e.g. multiple locus at the same time. The first plamid (pCas9) does express Cas9 plus tracrRNA and crRNA already (the natural version of gRNA, gRNA is a synthetic chimeric RNA of tracrRNA+crRNA), if I interpret the figures correctly. The second plasmid only provides more crRNAs for multiple targeting. Other possibility: the Cas9 decribed there is a Cas9 Nickase, so you have to use 2 gRNAs or 1 tracRNA + 2 crRNAs to target sites.

Thanks. Your explanation makes a lot of sense.

The non-profit plasmid repository Addgene has a page on resources for using CRISPRs in bacteria.

http://www.addgene.org/CRISPR/bacteria/

Perfect timing, I read the page and ordered the plasmids. Thanks.

Thanks for sharing your experience. px458 is mammalian expression, I guess

Thanks