I am trying to perform a gene knockout using pCRISPR/pCAS9 plasmids. Do I need to insert guide into pCAS9 plasmid for this purpose or just transforming the pCAS9 into the organism will be adequate?
Without a guide, the nuclease will not target your gene. So yes, you need a guide.
Thank you for the suggestions Lech and Antonio.
While I understand that I need to insert a guide in pCRISPR plasmid, I have not been able to wrap my head around inserting a guide in pCAS9 plasmid. So I have to insert guides to both pCRISPR and pCAS9 plasmid in order to induce a gene knock out?
As far as I know CRISPR consist of the Cas9 and the gRNA component. If you have a plasmid which can express the gRNA and Cas9 then it will work I guess. I use pX330-U6-Chimeric_BB-CBh-hSpCas9 plasmid where we can insert our gRNA and Cas9 is expressed from addgene and it works really well in my knockout experiment.
Dear Antonio Ruiz-Vela and all,
Thanks for giving the paper regarding this issue. However, in this paper they said that two plasmids are required because a pCRISPR plasmid containing a spacer targeting the E. coli chromosome cannot be constructed using this organism as a cloning host if Cas9 is also present (it will kill the host).Whould you please explain it, Why particularly in case of E. coli, the construction of the CRISPR-Cas system is not possible if this organism is also used as a cloning host? Please tell me the details.
I am going to use the CRISPR/cas 9 system for editing my host bacterial genome. and want to use only pCas9 plasmid for new spacer cloning without pCRISPR plasmid. Can I knockout the bacterial gene if i clone the spacer sewuence only in pCas9 plasmid?
can you tell me please which transfection reagent you used for transfection of pX330-U6-Chimeric_BB-CBh-hSpCas9 plasmid Lalhaba oinam
Dear Heba,
Lipofectamine 3000 from Thermo Fischer works for me and gave me good transfection efficiency.
https://www.thermofisher.com/jp/ja/home/brands/product-brand/lipofectamine/lipofectamine-3000.html
Dear Lalhaba
I used the same transfection reagent for MCF-7 KCR cell line but I got negative results,can you tell me the procedures that you used.
also I would like to ask you how you measure the transfection efficiency by FACS for example because this plasmid not contain GFP ?
Thanks in advance
Hi Rajan,
How did you finished your knock out by using pCas9 and pCRISPR? I experienced the same issue as you.
I also do not know whether to insert the protospacer into pCas9 or pCRISPR or both. Another question is which E.coli host did you use for knock out? Thank you very much.
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