CRISPR/CAS transfection, nucleofection

[Paul]

Hi,
I can't really find a protocol for the tranfection or nucleofection of CRISPR/CAS.
I have got the px330 vector.
How do you do the transfection of the plasmid (I have got a nucleofector)?
How much plasmid-DNA do you need for how many cells?
Do I have to consider anything in particular?
Thank you very much!
Paul

[sqj]

 try to find some reagents from this company

 

Ez biosystems

http://ezbiosystems.com/Resourcesview.asp?d_id=12

http://www.bio-world.com/productinfo/1_879/139436/AR-J-Cell-Avalanche%E2%84%A2-Transfection-Reagent.html

http://ezbiosystems.com/view2.asp?d_id=234&Name=AR42J%20Cell%20Avalanche%E2%84%A2%20Transfection%20Reagent

Cell Type-Specific Avalanche™ Transfection Reagents

[Paul]

thank you, but we have a Nukleofector.
I juyt would like to know how much DNA you take for how many cells or if you maybe transfect mRNA instead of DNA.

[Fei Ann Ran]

Hello,

Lonza provides various nucleofector kits for different cell types:

http://www.lonza.com/products-services/bio-research/transfection/nucleofector-kits-for-cell-lines.aspx

Lipofectamine 2000 (Invitrogen) also works for many cell types.

For both, we typically use 0.5-1ug of total DNA. For nucleofection, transfect 200,000 cells, and for Lipofectamine, plate 100-150,000 cells one day before transfection.

-- FAR

On Wednesday, June 12, 2013 9:28:22 AM UTC-4, Paul wrote:

[Chris Richie]

@Fei Ann Ran, 

Could you please elaborate on your protocol for using Lipofectamine 2000?

I am specifically interested in the following:

what cell types have you tried?

What is the ratio of the DNAs used for transfection (Cas9:guide RNA:Donor template)?

What is the plate/well format used when you are plating 100-150,000 cells ?

Thanks

Chris

[Fei Ann Ran]

Hi Chris,

We've used Lipofectamine 2000 with HEK 293FT/293T, N2A, and Hepa1-6 cell lines, following their standard protocol (for 24 well plates, 500ng of DNA total in 50ul of OMEM, with 2ul of Lipo).

In general a 1:1 molar ratio has worked well for Cas9:sgRNA. For ssODN donor templates, we've been using ~10uM per 24-well (see below); alternatively, one could use 400ng of plasmid donor template.

We typically plate 100-150K cells into each well of a 24-well plate, and transfect 18-24h later with a total of 500ng of combined Cas9 and sgRNA.

Ann

[Chris Richie]

Thanks for your fast reply!

This is going to be very helpful, since I have see a lot of posts describing nucleofection, but not much dealing with lipofectamine.

I intend to use pX330 to express the sgRNA and the Cas9, so they are already as equimolar as I can make them. 

The lipofectamine 2000 protocol suggests a maximum of 800ng plasmid DNA for each well in a 24-well plate.

Could you clarify your amount of ssODN?  Do you mean to include the oligo at 10uM (final) in the transfection mix along with the Cas9/sgRNA mix?  Is this amount of ssODN added on top of the 500ng plasmid Cas9/sgRNA component?

If using a plasmid donor template (at 400ng per well in a 24-well plate), is this added on top of the 500ng of Cas9/sgRNA components? 

To put it another way, does the inclusion of the donor template (oligo or plasmid) necessitate any reduction in the amount of Cas9/sgRNA plasmid?

Thanks again!

Chris

[Yilong Zou]

Hi Ann, thanks for sharing your protocol! May I get a clarified info about the "10uM ssODN donor template", is 10uM the final concentration of ssODN in the transfection mix? Or is 10umol the total amount used for each transfection? Thanks again,

Yilong

[Patrick David Hsu]

Hi all,

Sorry for the confusion. We typically add 1 uL of 10 uM ssODN (single-stranded donor) per well of a 24-well plate, so the exact nanogram amount will vary depending on the length of your ultramer.

This is added on top of the Cas9/sgRNA total; this may indeed add up to >800 ng (as recommended by the Lipo-2000 protocol), but the transfection still works very well.

Patrick

[lili...@gmail.com]

Hi tom, did you find publication about transfect Cas9 mRNA into cell?  not microinjection.  Thanks  li

[Neuropath]

A detailed protocol is given in this publication
http://www.ncbi.nlm.nih.gov/pubmed/24157548

[Jerry Xu]

Still confused:

Ok to use lipofectamine 2000 or 3000 for the transfection of ssODN?

Someone said only nucleofection is effective for ssODN.

[Tao Ye]

I have the same question. For HDR by ssODN, is it OK to use lipofectamine 2000 or 3000? One paper used X-tremeGENE 9 (Roche). Most paper used Lonza nucleofection. Thanks for your instruction.

[Carter]

I previously contacted Promega (Viafect and Fugene HD) and Mirus Bio (X2 and LT1) about it. Neither company is sure about an oligo of this size. Mirus thinks X2 might work. Promega is going to test Viafect. Mirus sells an oligo transfection reagent they think will work for ssODN. You would make your oligo complex with this reagent. However, you would also need to make a complex for your plasmid using another reagent. Both complexes would be added to your cells. I previously asked a question on this forum regarding ssODN and if it would be ok to add a fluorophore to it so you can monitor the transfection efficiency. I am concerned that the fluorophore will impact HDR efficiency or possibly inhibit it. Any ideas?

[Jonathan Geisinger]

Any polycationic lipid complex or electroporation will work.   

The fluorophore idea would depend on the fluor and the location.  Biggest drawback would cost, probably.