Transfecting method for ssDNA

[Neal Amin]

Hello all,

Transfection of the px330 and px335 plasmids for CRISPR-mediated modification has been working well for me, however, whenever I try to transfect an ssDNA donor for HDR, my transfection efficiency drops tremendously and i cannot detect any HDR.   Ive been using IDT oligos (no PAGE purification, based on recommendations I read in this Google Group) with lipofectamine LTX. Ive been using half the weight of DNA as the px330 plasmid and the other half of the weight is ssDNA.

I am wondering if someone can share their protocol for transfecting ssDNA donors... answers to the following could help me: 

From where do you purchase the single stranded oligo? 

What kind of purification did you have ordered or performed yourself? 

Did you PCI extract the ssDNA before transfection?

Which transfection reagent do you use? 

What ratio between pX330 plasmid and ssDNA do you use?

Thanks in advance,

Neal

[p.pri...@gmail.com]

Hi Neal,

I can't rightfully answer the rest of your questions as I'm not yet at that step, but I've ordered oligonucleotides from Eurofins.  Now I ordered DNA for the intention of plasmid cloning, but they arrived as non-annealed ssDNA, perhaps that will work for you.  Here is the link:

http://www.operon.com/products/custom-oligos/getting-started.aspx

Best of luck,

Phil

[Betsy Gray]

Hi,

I've been having a similar problem.

I’m trying to use single stranded oligo donor templates to make defined mutations in 293T cells (e.g. introduce LoxP sites, etc). I’ve tried using the hEMX1 gRNA + donor ssODN described in Hsu et al (Nature Biotech, 2013: http://www.nature.com/nbt/journal/v31/n9/full/nbt.2647.html) as well as a few 160bp donor ssODN that were designed to introduce a LoxP site into my target gene (the LoxP sequence in the donor disrupts the gRNA target site, so my gRNA shouldn’t cleave the donor sequence). In each case, I do not see any HR, and introducing the ssODN actually inhibits NHEJ. Has anyone else had a similar problem? Any tricks for getting this to work?

A few specific questions:

(1)  A few people have recommended trying PAGE-purified oligos from IDT, although from other posts here it seems that isn’t necessary. Thoughts?

(2)  For 1e5 293T cells, I have tried transfecting ~200-2000ng of donor oligos (with Cas9 plasmid + gRNA) with 2ul Lipofectamine 2000 (should I use more Lipofectamine?). I prepare a 3uM stock of the oligos in water, and use 1-10ul of the stock for each transfection. Could this be causing any problems? Should I resuspend my oligos in something other than water?

Thank in advance for your help. Any suggestions would be greatly appreciated.

Betsy

[Patrick David Hsu]

Hi all,

For doing HR with single-stranded donors, nucleofection gives much higher efficiency than lipofection.

We use the SF cell line kit from Lonza, non-PAGE purified ultramer oligos from IDT, and a 1:1 molar ratio of Cas9:sgRNA (higher guide ratio works well too - 1:3 or 1:5). For a given nucleofection, I use 500 ng of Cas9 and 1 uL of 10uM single-stranded donor (variable in concentration depending on length of ultramer) for 200,000 cells. 

Please see our new protocols paper for details: http://www.nature.com/nprot/journal/v8/n11/full/nprot.2013.143.html

Best,

Patrick

[Manuel Kaulich]

Hi Patrick,

Thanks for the clarifying notes. Can you say what kind of %'ages of HR you are talking about for Lipo- or Nucleofection? Have you directly compared the two techniques (e.g. on the EMX1 locus)?

Thanks for your kind help.

Manuel

[Neal Amin]

Thanks for the input, everyone.  It might be that nucleofection is necessary to get ssDNA into cells and that lipofection doesnt work.  Ill have to give this a try, since its clear to me that something is wrong with my lipofection only when I use ssDNA.  

I will try the method in the nature protocol.

Neal

[Mingyi Xie]

Hi Neal, 

I will start doing a similar experiment. Before doing anything, I am checking online to see if there is anything I need to pay attention to. And I've come across your posts. Did it work for you? Neucleofection is better than lipofectamine in this case? Thanks.

[Marcos Sixto]

Hello Neal, did you ever get the HDR to work using SSDNA and lipofectamine?

[Rajiv]

Bump! Also curious if you noticed significant differences regarding HR template repair when using nucleofection. I know that for some cell lines transfection obviously works much better with nucleofection compared to Lipofectamin, but after selection you do the serial dilutions anyways only with actually transfected cells. So I am curious if you have better efficacy of repair with your template with nucleofection compared to Lipofectamine.

Thanks! 

[amrel...@vet.suez.edu.eg]

Hi, Amin 

please, can you tell me the length of your ssDNA and should be 5 prime end phosphorylated or not. 

Thanks