Deletion using two pairs of TALENs

[elph...@gmail.com]

Dear all,

I am wondering wether anyone has successfully used two pairs of TALENs at once in mammalian cells to induce a deletion of the DNA in between the two target sites. If so how big was the intended deletion(s)? Would it be realistic to think about a couple of kbs or more?

Many thanks for your help!
Best, Elphege

[Amir Taheri]

Dear Elphege,

It would be successful as I found similar matter in a US patent application "GENETICALLY MODIFIED ANIMALS AND METHODS FOR MAKING THE SAME" .

Good luck with your project.

Amir

[Amir Taheri]

Dear Elphege,

I thought it is not bad idea to test the strategy explained in a Nature Method paper which seems you would be able to delete a region by using a combination of TALENs and ssOligoes:

high-frequency genome  editing using ssdna oligonucleotides with  zinc-finger nucleases

Cheers,

Amir

[Amir Taheri]

Sorry for duplicate emails, also below article you may find it interesting regarding to make a big deletion in the genome:

Highly Efficient and Specific Genome Editing in Silkworm Using Custom TALENs

[elph...@gmail.com]

Hi Amir,
Thanks a lot for these info. I am trying all this right now but I was wondering whether anyone around here had already given it a go in mammalian cells at all.
So much still unknown!! Elphege

[Bisola C]

Hi Elphege,

 This is a paper where they worked with the same principles in human/mammalian cells. ZFN though, but i believe it would work for TALENS.

http://www.ncbi.nlm.nih.gov/pubmed/19952142

good luck..

 Bisola

[Davide Seruggia]

Hi!

Got to be really lucky ( =screen many clones) to get both TALENs cutting on the same allele! The 1 nuclease + ssOND (published with ZFN) avoid this problem.

best

Davide

[Stephen Ekker]

Not if you have effective TALENs. I would guess that one quality TALEN pair plus an average TALEN pair would be more effective that using ssONDs.

[sm20...@gmail.com]

Dear Elphege,
I am curious whether you were successful with your TALENs deletion experiment by now because I am planning a very similar approach. So I would be very grateful for any advice!

Best,
Matt

[elph...@gmail.com]

Hi Matt,
I have used two pairs separated by 50kb. If I prep DNA from the whole cell population 2d after transfection and I run PCR with primers flanking these 50kb I can clearly detect several deletion events. I do not know however how prevalent these are but I assume they must be pretty rare. I am growing clones now hoping to isolate some with the deletion. I am also trying with pairs of TALENs separated by smaller distances. I'll keep you posted on how it goes!

[Stephen Ekker]

We are seeing large deletions from two TALEN pairs in somatic zebrafish cells. Should work well in mammalian cells.

[sm20...@gmail.com]

Hi Elphege, hi Stephen
thank you for your comments. I am planning to delete ~20 kb. Did you guys also use donor template for drug selection or GFP sorting in that setting? To complicate things I am only interested in targeting one specific allele, which I believe will be challenging.

Best,
Matt

[Stephen Ekker]

Right now, we are only using TALENs without any donor templates for this work.

[elph...@gmail.com]

OK so I did the experiment with TALENs +/- oligos and here are my observations.

Cells transfected with two pairs of TALENs separated by 3kb: could detect deletion in the transfectant pool, though the band for deletant is really super weak compared to WT band. I am growing clones now but I expect frequencies way lower than 1/100...

Cells transfected with one TALEN pair and one ssOligo: no deletion and no insertion in the transfectant pool. Also tried an oligo for point mutation (no deletion) without success.

Conclusion: two pairs of TALEN was here more efficient to induce deletion than 1 TALEN + 1 ssOligo.

My question now is: has anyone here successfully used an ssOligo strategy to induce a deletion, an insertion or a substitution? I would like to persevere as oligos would obviously be super convenient to use! Thanks in advance for your comments! Elphege

[Stephen Ekker]

Great note, lots of questions:

1) Obviously, the efficacy of your TALENs is a big deal as it limits the number of modified chromosomes. If you are only seeing 1% or less of breaks for your TALENs via NHEJ, then adding the relatively inefficient oligo as donor is not going to show much activity. Also, the design of your oligos is very important. At least in zebrafish, shorter oligos (75mers, for example) work better than longer oligos (100mers).

2) We have used oligos to make small changes and insertions. We have not tried to make big deletions using an oligo as donor plus a single TALEN pair.

[Davide Seruggia]

hi everybody,
I'm working on deletions with two TAL pairs.
when I transfect two pairs plus GFP, I observed that two tals result quite toxic (decrease in GFP pos cell) compared with single pair (transfection topped up with pUC to use same DNA amount).
the difference is even stronger 8d post transfection. I use 400ng per TAL in 24well plate, with Lipo2000. Do you see the same?

best
Davide

[eddie-...@gmx.de]

Thought about a lethal effect caused by the big deletion occuring through the usage of your two TALENs? How big is the genomic region you remove?

I guess it doesn't need to be a toxic effect by itself....

Best
E

[Davide Seruggia]

Hi Eddie,

I'm deleting a 1.5kb sequence. KO cells and mice of the gene I am targeting are ok, so I don't expect the deletion to be lethal itself. Rather, I thought that the cells I am using are ''bad recombinators'', can't recover after DSB and die. I could detect the deletion at day 3 post transfection, but the drop in GFP+ (cotransfected with the TALs) cells at day 8 may indicate that I am loosing the targeted cells. Maybe the continuos expression of TALs is indeed toxic? I am using pCAG-T7-Sangamo vector in mouse Neuro-2A cells.

best

Davide

[sm20...@gmail.com]

Hi Elphege,
Can you tell me where you bought your ssODN?
Best,
Matt

[elph...@gmail.com]

Hi,
I purchased PAGE purified oligos from Sigma-Aldrich. Again, my attempt was unsuccessful (with TALENs than I know are cutting). Let me know if you get it to work!
Cheers

[Stephen C. Ekker]

The experimental design of the ssODN is going to matter, as well as the ratio of ssODN to TALEN and how you are accomplishing gene transfer.

The two TALEN pair approach is only really a function of having two working TALENs, and you will likely be co-delivering those.

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[Eliška Svobodová]

Hi,
I've achieved to generate mice with 6 Kb deletion using 2 pairs of TALENs with no oligo. Efficiency of TALENs in cells (NIH3T3) was quite low and detection of deletion was very weak. When TALENs were injected into oocytes for generation of KO mice, efficiency was 3/60. I'm now planning to use 2 different TALEN pair together with oligo for similar deletion but I need to have precise sequence at site of deletion/junction of two arms.

Why is it better to use ssOligo than ds DNA?

Dne středa, 17. října 2012 22:42:22 UTC+2 elph...@gmail.com napsal(a):

[Stephen C. Ekker]

Hi, Elphege.

A few comments:

1) The TALENs you are using seem to be less than optimal for your application. They may become a bottleneck whether you use an oligo or dsDNA as a donor.

2) ssDNA is normally better at making SNP-type edits; dsDNA for larger genome changes. In rodent embryos, it might be better to use dsDNA as a donor in your applications. One real advantage of ssDNA is that you just have it synthesized. dsDNA donors tend to take several weeks to make in the lab.

Hope these answers help!

Steve

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Stephen C. Ekker, Ph.D.

Director, Mayo Addiction Research Center

Professor of Biochemistry and Molecular Biology

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Mayo Clinic Cancer Center

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[banni...@gmail.com]

Hi All,

Lots of comments so far, but just wanted to add that we often use two pairs of TALENs targeting distal exons of the same gene and get large deletions (so far up to 1.5kb) with varying efficiencies (depending on how efficient the TALENs are!)

We haven't used ssODNs to bridge the gap so to speak, but I recall seeing this reported in a few papers (also with CRISPR/Cas9) so it should be possible, but you need to have really efficient TALENs in the first place (for both sites) for this to work well!

cheers
Steph