Cloning into PX458 plasmid

[Cosmas Giallourakis]

Hello all

I am very new to the crispr field so forgive my questions

For the PX458 plasmid I am trying to figure how to clone a guide into it and have few questions

1) There are 2 BbsI sites (why two?)

2) Is there already a guide RNA that I have to remove before cloning into the Bbs1 site?

3) Which protocol (can I have link) do I following in terms of designing oligos and there overhangs

Thx for the allowing me ask perhaps these very simple questions

best

Comas

[Kim]

Hi, 

The protocol for cloning can be found in Ran et al, Nature Protocols 2013, Genome engineering using the CRISPR-Cas9 system (http://zlab.mit.edu/assets/reprints/Ran_FA_Nat_Protoc_2013.pdf). There, they also explain about the use of the BbsI restriction sites. Hope this helps :)

Op vrijdag 4 april 2014 13:29:15 UTC+2 schreef Cosmas Giallourakis:

[dorina avram]

you clone in the same BbsI sites and use the same strategy if you use the pX461, right?

[Emma]

The 2 BbsI sites are in the opposite orientations and produce 2 cuts flanking the 2 recognition sites of the BbsI enzyme. This has 2 advantages: 

1) Excision of the BbsI sites upon cleavage and gRNA insertion. This prevents re-cleavage and can help with assessing success of the gRNA insertion (by RE digest).

2) The opposite orientation of the sites mean that the 2 overhangs do not match so your plasmid is not likely to ligate to itself and will instead match up with your gRNA overhangs. 

There is no gRNA that you need to remove, your gRNA will replace the bp between the 2 BbsI sites.

I hope it's working well for you :)

[Duško]

so,if i understand u: the cloning technique into px458 vector is the same as cloning into px330.Just ligate two primers (after anniling) with appropriate overhangs for BbsI overhangs?
and the result will be fluorescence in cells after Cas9 cutting?

thank you for your answer

best

Dne petek, 25. april 2014 21:19:56 UTC+2 je oseba Emma napisala:

[JP]

All transfected cells will be GFP+. Not all transfected cells will have indels.

[tomasz.u...@gmail.com]

Hello

I have a bit of similar question. I'm trying to figure out why it is possible in this protocol to perform BbsI digestion and oligo ligation in the same reaction mix. It's in the protocol on page 11. How come there is no plasmid gel purification step and then ligation? Is there no chance that BbsI sites will re-ligate into the plasmid? Please help, I'm running circles here. Later on there in step 5 vii there is digestion of linearise residual DNA but it is after ligation so have nothing to do with plasmid - BbsI sites sequence re-ligation.

Cheers
Tom

[Tim Vierbuchen]

As stated before, the 2 recognition sites of BbsI are on the smaller fragment that is cut out but not on your backbone vector. It's a type IIS restriction enzyme that cuts outside of its recognition sequence on one side. Therefore, the enzyme will readily cut the "old" insert out if it religates. The "new" insert (coding your gRNA sequence) won't be cut out as it doesn't contain BbsI restriction sites.

Does this answer your question?

Best
Tim