Genome editing tools, gene deletion?

[Koeng]

Hello all!

I was just checking out some stuff about genome editing, but I couldn't really find anything about gene deletion inside a genome. Perhaps using a TALEN, would work is what I was thinking

A system goes like this: (the site it attaches to is also in the NNNNNNNN part)

ATATATAT'NNNNNNNN'GCGCGCGC
TATATATA'NNNNNNNN'CGCGCGCG


Then it is cut

ATAT                   CGCGGCGCGC
TATATATA                      CGCGCG


And I add some DNA

ATAT'N
         N'GCGC

So it ligates

ATATATAT 'N' CGCGCGCG
TATATATA 'N' GCGCGCGC

Or what would the Bacteria do if its genome got cut like this? Die? Any documents for me to read would be helpful, or just your opinion. Thanks!

Koeng

[Mega]

Hi!

That looks good, the insert you painted has the neccessary recognition sequence so it will attach to the target and insert.

Basically it would be blunt ended then, right? What if you design your insert like ... No it is not blunt ended because your  top insert N base-pairs with the bottom insert N. 

The cell would surely recognize this as a strand break, and thus DNA ligase would ligate it. So I guess, as long as DNA ligase comes quicker than an exonuclease,   the bacterium will survive. Most probably in the major part of the cells... 

[Mega]

But gene deletion should be much easier with miRNA, if I'm correct there. It doesn't need a targeted integration, just any place is valid. 

[Cathal Garvey (Phone)]

Splicer complexes like miRNA antisense systems are fairly exclusive to RNA, I think, although you could probably (if so, someone may have already) hack it otherwise.

To selectively excise a chunk of DNA while closing the ends neatly, you could use a integrase/excisionase system, but for the the flanking sequences and resulting 'scar' site must be predefined according to the enzyme.

There's a class of restriction enzyme that excises a chunk of target DNA,sometimes without requiring or leaving boundaries, but they don't repair the free ends. Fusing one to a ligase might help by ensuring that DNA only gets excised when a repair enzyme is close at hand.

Mega <masters...@gmail.com> wrote:

But gene deletion should be much easier with miRNA, if I'm correct there. It doesn't need a targeted integration, just any place is valid. 

--
Sent from my Android phone with K-9 Mail. Please excuse my brevity.

[Koeng]

Class 2 I believe? Fokl is a class 2, and the site it binds to can be engineered (TALENS or Zinc Finger). So hypothetically if I add a ligase to it, they will cut the dna then ligate it together? The problem I am thinking of us if I attach a ligase, the Fokl enzyme will cut then the ligase will ligate the stands back together! Would it be possible to ALSO attach a enzyme to blunt the sticky ends so then the 2 ends of the chromosome will be able to ligate together?

I checked out miRNA too :) it seems pretty cool, but the only problem is I am not looking for gene silencing, but entire gene deletion. I was thinking of perhaps using this to created a minimal bacterium by using a plasmid, each with synthetic TALENS to delete one gene. But that is just an idea :)

-Koeng

[Josiah Zayner]

There are lots of easy ways to do genome editing in Bacteria mostly using homologous recombination. A common one is recombineering http://en.wikipedia.org/wiki/Recombineering . Another is through conjugation.