Hello Roshan,

sgRNAs can be multiplexed by HiFi or Gibson. However you cannot use the scaffold region to assemble them, as it will be identical and the position or number of the sgRNAs you'll try to assemble will vary a lot. I am introducing small unique sequences between the cassettes to assemble them.

I have assembled up to 4 hU6-sgRNA-scaffold cassettes as tandem repeats using Gibson or HiFi. Alternatively I am also assembling polycistronic sgRNAs cassettes using self-splicing Gly-tRNA sequences.

Regarding the second strategy you can have a look at this paper we published:

Article (Po)STAC (Polycistronic SunTAg modified CRISPR) enables live...

Although for this work the sequences were synthesised you can have an idea of the cassette structure. To assemble this kind of cassette by PCR and HiFi however you will need the original templates.

Hope it helps

Francesco

Hi Roshan,

why don't you simply order a synthetic DNA sequence? Gene synthesis has become so cheap we completely stopped assemblying DNA segments. We have recently published a paper where you can see in the supplementary figures how we design multiplex sgRNA expression casettes for a gene synthesis order (PMID: 29451908).

Best,

Leon

Hi Roshan,

why don't you simply order a synthetic DNA sequence? Gene synthesis has become so cheap we completely stopped assemblying DNA segments. We have recently published a paper where you can see in the supplementary figures how we design multiplex sgRNA expression casettes for a gene synthesis order (PMID 29451908).

Best,

Leon

sorry, didn't want to spam you - there is currently an error in the researchgate input screen.