pX335 vs pX461 versions of pSpCas9n(BB)?

[Adam Cook]

Hello,

A colleague in my department already has pX335, but I notice from Ran et al 2013 (Nat Protoc) that there is a more recent version of 'pSpCas9n(BB)', pX461 (also available on Addgene). Is it recommended that I obtain pX461 from Addgene for double nickase, or should pX335 be fine? I notice by alignment that there only appears to be a very minor difference with an NLS sequence, aside from swapping the HA tag for FLAG.

Thanks in advance!

Adam

 

[Le Cong]

Hi Adam, the major difference is that pX461 have the 2A-GFP inserted after the SpCas9 gene so you will be able to visualize the cells bearing this plasmid using GFP fluorescence. This can come in handy when you try to perform selection/sorting of transfected cells to increase the yield of genome editing. The components of these two plasmids should function essentially the same in cells since other differences are minor changes.

Best,

Le 

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[Adam Cook]

Hi Le,

Thanks for your response. Actually I meant to type pX460 (without GFP), not 461. 

I'll proceed with pX335.

Adam

[Fei Ann Ran]

Hi Adam,

PX335 and PX460 are more or less equivalent. PX460 uses the same NLS as PX330 and PX458-PX462, whereas the PX335 plasmid contains a differently codon-optimized NLS sequence. This was standardized with the PX460 series to standardize cloning and primer designs for modifications.

Best,

Ann