I do not have this experience yet, but It is exactly what I am going to do. In my case I am doing a gene knock-out. I requested (and received) a gRNA design for my gene of interest (GOI) from a Company that I was planning to use. The CRISPR technology has revealed to be much more challenging than how it is advertised: for one single gRNA CAS9 will chop 7 to 15 unspecific sites in the genome. And think that the Company suggests to use at least 4 gRNA per one extremity of my GOI. Considering that there are 2 extremities, at least 50 off-target cuts will be produced per one my GOI. In other words the genome risks to be chopped in pieces.

Thanks for you answer, this sounds really challenging...genome chopped into pieces couldn't be easy to handle, even not for selfrenewing stem cells. damn it! So would it be possible to tell me which company you have chosen? in a cooperating working group at my university they already use CRISPR and i will meet them next week. if you like, i can keep you up to date.

wish you all the best with your project!

Sara

We got gRNA design from Invitrogen. However, the off-target cut is not something "Company-wise", it is about how the system works. gRNA is generally around 20bp long. Genome is full of nucleotide sequences with high degree of identity for the same sequence of gRNA. The trick is the selection of expanded clones, hoping that among them will be one with a single cut: that of your GOI :))

Yes, I would be grateful if you could let me know what they say about CRISPR experiment.

Best wishes,

Anastssia

Ya it is true that the off target effect is one of the major draw back of the CRISPR technology but still it is working fine in most of the cases. In my lab we have developed several KOs in different cell types. Even multiple gene KOs in a single cell type simultaneously. But sometime the cells are doing fine while put on selection but the phenotype is different. May be because the lenti virus is integrated into the genome but the gRNA is not doing its job. At the moment, I am trying developing KOs in Neutrophils which are very diificult to transduce and therefore the overall efficiency is quite low. Hope it is going to work....

Best of luck

Thanks

Thanks Prafull. I wish you all the best for your project too.

We tried one of the recent kits from Santa Cruz with our mesenchymal stem cells... do not try it did not work and is only troublesome!

thank you benjamin!

It looks like we're going take the plasmids from Addgene. Especially the constructs from the Zhang Lab. they have interesting modifications in Cas9, e.g. a Cas9 Nickase. They also have a gRNA prediction tool where you can see predicted off-target effects and if they are located in coding regions or not. Maybe that would be interesting for you.

Wish you all the best for your CRISPR experiments.

Yours Sara

http://www.addgene.org/crispr/zhang/#cas9

Hello, 

How did your CRISPR/Cas9 experiments in MSCs work? Are these cells easy to work with and amenable to gene editing? 

Thanks, 

Ralitsa

Hello Ralitsa,

I did not started CRISPR/Cas9 experiments in MCSs because we decided to focus on other experiments for now.

Working with MSCs and gene editing is not that easy with MSCs. If you have special questions you can send me a private message.

Yours Sara