Minimum distance between 2 gRNAs on same strand to achieve cutting?

[Olli]

We know when using Cas9n that we need to pay attention to the off-set in order to avoid steric hindrance of Cas9n protein binding to opposite strands. Are there any reports on what is the minimum distance required to achieve cutting with 2 gRNAs on the same strand using Cas9 wt? 

[ASH 1338]

Er, why do you want to cut the same strand? DSB only occur if the cut are on different strand.

For nickase on separate strand, spacing of down to 4nt (between 1st nt of gRNA next to PAM) can be efficient. If <4nt, then PAMs (NGG) will overlap for both nickases so the minium spacing is tolerated.

I think close spacing will still allow cas9 to edit but less efficiently. One Cas9 can bind, cut and  dissociate. Then NHEJ will happen and the first one no longer can bind. Then the other one can come in. Then you will have 2 edits in close proximity.

[ASH 1338]

I forgot to state source : http://zlab.mit.edu/assets/reprints/Trevino_MethodsEnzymol_2014.pdf (4nt spacing)

[Olli]

I intend to use 2 gRNAs (simply to increase the chance of creating a DSB in case one gRNA is not cutting) with Cas9 wt and was wondering what is the minimum distance between the gRNAs on the same strand to enable cutting. We know that Cas9 stays for hours after cutting DNA so I don't think you will get cutting with the other gRNA if too close to each other - hence how far should both gRNAs be apart to guarantee cutting?

[ASH 1338]

If you are using guides on the same strand, then steric wise i think you should start from >23bp. This will correspond to (N20)NGG(N20)NGG. Any closer and the first gRNA will prevent Cas9 binding and this will prevent tandem cutting.

But that is for tandem cutting. For non tandem cutting like I said, one cas9 can cleave after another. Cas9 stays for hours but cas9 plasmids (or are u using RNP?) will stay for days so there is still a significant chance the 2nd one will cut after the first one leaves.

[Olli]

Thanks for your input. Yes, I am using Cas9 protein.

[ASH 1338]

I think you can deliver the 2 RNPs in series. One after another. This will guarantee ``both`` cuts even if the site totally overlaps.

Are you using lipofection or electroporation, and do you observe signficant cell death? Perhaps this may be complicated if you use a lethal method like electroporation.