Including the PAM sequence in the gRNA sequence?

[omer gilan]

Hi everyone,

I was wondering what would happen if I included the PAM sequence in the gRNA vector, so instead of the 20bp guide sequence it will be 23bp, will that still work with cas9 to induce DSB?

Thanks in advance

Omer

[mutant bass]

Omer,

If the PAM you included in your vector isn't followed by another nGG or nAG triplet, it likely won't work. 

If the PAM you included in your vector IS followed by another PAM, it will likely work but not as efficiently as a 20-mer guide.

[omer gilan]

Hi, 

do you mean if the PAM i included in my vector isn't followed by another PAM in the genomicDNA? or followed by another PAM in the gRNA vector? 

thanks for your help,

Omer

[Hanna Kok]

As far as I know your PAM site shouldn't be in the guide, only on the genomic sequence. If you include it in your guide I assume Cas9 will cut your vector. I am not sure though!

[Hanna Kok]

See Page 2: https://www.addgene.org/static/data/plasmids/52/52961/52961-attachment_B3xTwla0bkYD.pdf
"Example oligo design: Note that the NGG PAM is not included in the designed oligos."