Hi All,
Have anyone try KO a gene from mammalian cell line with Cas9 protein + vectors that expressing gRNA?
Also, for generating KO clones, is it better to use protein-based or DNA-based CRISPR system? Which one will have better efficiency??
Thanks,
Lyra
Hi Lyra,
I have used the Cas9 with gRNA vectors. I am not sure what you mean by protein-based system, as nearly all of the methods for this technology that I have seen employ DNA vectors or other nucleic acids. For CRISPR/Cas9 you need two essential components: (1) Cas9 enzyme encoded by an appropriate mammalian expression vector and (2) gRNA either encoded by a mammalian expression vector or supplied as synthetic RNA. You can introduce these into the cells of interest in a few different ways, the most efficient of which will depend on what works best in your cells vs the trade off of time invested in the experiment. I have used both lentiviral based all-in-one vectors (i.e. one plasmid contains both Cas9 and gRNA, as well as a selection gene such as puromycin resistance) and co-transfection with individual components. I hope this helps you understand the system better.
In designing your experiment, it is important to also consider how you will screen your clones. Selection will help you out a lot, either through negative selection using drug resistance or positive selection such as cell sorting for GFP. Also having a convenient way to pick clones is nice. I settled on seeding my cells in 96 well plates after puromycin selection at 0.5 cells per well to minimize chances of wells with multiple clones, and used microscopy to confirm wells with single colonies. I also had some trouble with screening my clones. Initially I tried the T7EI enzyme method, but had very inconsistent and non-specific digestion problems. Then I stumbled upon a much simpler method that creates the same heteroduplexed PCR product and runs it on a TBE PAGE gel, where imperfectly duplexed DNA (i.e. mutation) migrates more slowly. (For a reference using this technique, see http://www.ncbi.nlm.nih.gov/pubmed/23075543)
Hope this gets you started.
Hi Lyra,
I have used the Cas9 with gRNA vectors. I am not sure what you mean by protein-based system, as nearly all of the methods for this technology that I have seen employ DNA vectors or other nucleic acids. For CRISPR/Cas9 you need two essential components: (1) Cas9 enzyme encoded by an appropriate mammalian expression vector and (2) gRNA either encoded by a mammalian expression vector or supplied as synthetic RNA. You can introduce these into the cells of interest in a few different ways, the most efficient of which will depend on what works best in your cells vs the trade off of time invested in the experiment. I have used both lentiviral based all-in-one vectors (i.e. one plasmid contains both Cas9 and gRNA, as well as a selection gene such as puromycin resistance) and co-transfection with individual components. I hope this helps you understand the system better.
In designing your experiment, it is important to also consider how you will screen your clones. Selection will help you out a lot, either through negative selection using drug resistance or positive selection such as cell sorting for GFP. Also having a convenient way to pick clones is nice. I settled on seeding my cells in 96 well plates after puromycin selection at 0.5 cells per well to minimize chances of wells with multiple clones, and used microscopy to confirm wells with single colonies. I also had some trouble with screening my clones. Initially I tried the T7EI enzyme method, but had very inconsistent and non-specific digestion problems. Then I stumbled upon a much simpler method that creates the same heteroduplexed PCR product and runs it on a TBE PAGE gel, where imperfectly duplexed DNA (i.e. mutation) migrates more slowly. (For a reference using this technique, see http://www.ncbi.nlm.nih.gov/pubmed/23075543)
Hope this gets you started.
Hi Jessica,
I think by protein-based system she is referring to: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4032847/
Hendrik,
Thanks for the reference. I was not aware that this approach was being used. Do you know how accessible this technology is (i.e. using common molecular biology techniques, commercial availability)? It seems like it would greatly improve on the current limitations and caveats of the CRISPR/Cas9 system, but could require some prohibitive biochemistry.
Jessica,
Thermo Fisher Scientific for example offers the Cas9 protein: https://www.thermofisher.com/order/catalog/product/B25640 and additionally a specific lipofectamine: https://www.thermofisher.com/order/catalog/product/CMAX00003
Hi Jessica,
Thank you so much for your reply!
I know that you can express & purify Cas9 easily from E.coli because its a bacterial protein. You can get the expression vector from Addgene (https://www.addgene.org/62934/).
What I mean by question is that:
I know you can use (1) mammalian vector expressing Cas9 & gRNA, or (2) Cas9 protein + commercial synthesized gRNA.
However, if I don't want to buy gRNA from company, can I just transfect the cells with purified Cas9 protein and mammalian vector that express gRNA? I am just curious about if anyone tried it before.
I have heard people said that the Cas9/gRNA system is more specific & efficient than the plasmids.
After searching/reading literature, I decided to use the vectors from Zhang Lab. I am planning to try out three of their vectors: PX458, PX459, and eSpCas9(1.1). I will test them out and let you guys know how it goes.
Thanks,
Lyra
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