THP-1 CRISPR protocol

[Bryan]

Using the protocol outlined below, I am unable to detect integration of our donor construct by PCR or GFP expression in THP-1 cells. Does anyone have a protocol or a reference to the use of recombinant Cas9 protein in THP-1 cells using nucleofection, or a suggestion to improve our current protocol? THP-1 cells are recalcitrant to cationic- and lipid-based methods of transfection, meaning I was unable to use a Lipofection protocol.

Protocol for recombinant Cas9 NLS Nuclease protein to edit the genome of a THP-1 cell line:

1. Add 1 ug of Cas9 nuclease preloaded with 250 ug of validated (in HEK293M) sgRNA to form the ribonucleoprotein complex. 

2. THP-1 cells are electroporated with 100 ng of endotoxin-free linearized donor DNA (a homology-directed repair template) using an Amaxa Nucleofector IIb with protocol U-001. 

3. The cells were cultured for 96 hours in 1 uM SCR7, a NHEJ inhibitor. 

I know THP-1 cells and monocytes in general are deficient in HDR, so any help to optimize this is appreciated!

[Ancheto]

Why do you say THP1 are deficient in HDR ("in general")?

Ana

[tlmo...@uvm.edu]

Hi Bryan,

I was wondering if you ever got CRISPR to work in your THP-1 cells. 

I believe monocytes are in general deficient in many components necessary for DNA repair making CRISPR difficult. See here: 

Human monocytes are severely impaired in base and DNA double-strand break repair that renders them vulnerable to oxidative stress

Martina Bauer, Michael Goldstein, Markus Christmann, Huong Becker, Daniel Heylmann, and Bernd Kaina¹

They also upregulate cGAS-STING such that cytosolic DNA triggers cell death if you use a plasmid mediated approach.

Did you find a protocol that works despite this?

Theresa

On Tuesday, August 16, 2016 at 7:01:50 PM UTC-4, Bryan wrote:

[Isa Ferraz]

Hi Bryan

Please let mw know if you could edit the cells, becouse I´m trying to do the same protocol.

Thank you.

Isabela

[Nikhil Madhusudhan]

Bryan - The first think I would do is make sure you can transfect the cells (with the nucleofector). Just do a few experiments where you optimize transfection. Use a GFP plasmid such as pMAXGFP to determine quickly if transfection has worked. Once you have done that you can use a plasmid like px330 to introduce Cas9 plus sgRNA into cells. You can co-transfect you donor DNA template along with this as well. 

Another thing I would do before transfecting the sgRNA and DNA repair template is check how good your guide cutting efficiency is by surveyor. You can do this in 293T cells to make life easier, but it is best to do it in the THP-1 cells. Test a few different sgRNA's that will cut at your required site and then make sure that you go forward with the guides that cut well.

The thing is right now you don't really have any evidence that your guide even works or that you are getting successful transfection. Once you can get good transfection and get good guides that cut, then you can worry about genotyping your edited cells.

[Niraj Shah]

Hello, my lab have used crispr cas9 in thp1s.
As mentioned by others- they are hard to transfect cell lines, in the end we used a lentiviral transduction with the LentiCrisprv2 vector. I should note, that even with puromycin selection the editing efficiency was a lot lower then most other cell lines we have previously used.

In theory I cannot see why an RNP based method wouldn’t work in these cells

[Katherine Cummins]

Yup THP-1s are easily editable (for knock out of a gene) using an RNP / nucleofector appreoach. The Lonza 4d worked great for me, and they have a publically avialable protocl including their recommended pulse code, and electroporation solution. I used CRISPR/Cas9 and IVT gRNA delivered as an RNP to the cells while in suspension.

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