U6 or T7 promoter on PX330 vector

[tony zeng]

Dear all, I am new in this field. I read the paper "One-Step Generation of Mice Carrying Reporter and Conditional Alleles by CRISPR/Cas-Mediated Genome Engineering" by Hui Yang, et al. Cell 2013.

They added T7 promoter to the Cas9 coding region of vector PX330 , purified it for in vitro transcription and then did the same steps to the sgRNAs template of vetor PX330. there is no problem for it. However when I went to website of addgene (http://www.addgene.org/42230) and found that PX330 has a U6 promoter already in the beginning of vector and then gRNA insertion site and hspCas9.

for my experiment, I want to produce Cas9 mRNA and sgRNA separately and then micro-inject them with oligos into mouse 1-cell embryo. However, for addgene PX330 vector, how can I get Cas9 mRNA, sgRNA by separately? Can you recommend me suitable vectors and protocols? I just want to make my experiment as simple as possible.

Thanks

Tony

[Rajat Singh]

The pX330 does not have the T7 promoter. You have to amplify the sgRNA template (the one you cloned into the BBSI site of pX330) from the pX330 plasmid using a primer that has the T7 promoter at the 5' end and a common reverse primer for pX330. 

Forward primer: T7 promoter (19 nt) + BBsI site CACC (4nt) + 20/21 nt targeting sequence == ~44nt primer.   

Reverse primer is AAAAGCACCGACTCGGTGCC. 20 nt long and is common for all sgRNA from pX330. 

The amplified template will be around 120 bp.

You then use a T7 promoter IVT kit to make the sgRNA. 

I am not sure if you can do the same thing for the Cas9mRNA as I don't know how big Cas9 is (we are using a core facility for injections and they make the Cas9 mRNA for us). 

[tony zeng]

Thank you so much! Rajat,

I will contact with core facility here to see if they can provide Cas9 mRNA for use.

Tony

On Wednesday, August 6, 2014 11:46:24 AM UTC-4, tony zeng wrote: