Changing PAM sequence for HR repair

[Anoeska]

Hi,

I want to introduce a point mutation, therefore have designed sgRNA's that will lead to a cut around the mutation site.

The repair template will be a single-stranded DNA oligo, with ~50 bp homology arms and including my desired point mutation. So far, everything seems great.

I will have to change my PAM sequences in the repair, else the repaired DNA could be cut again and again and efficiency might become quite low. So it's not that the repair ssDNA is cut (it is sense to the sgRNA so won't bind) but I am worried that my repaired DNA will be cut after repair.

Unfortunately, changing NGG to NGT or NGC does not give a silent mutation. Same for NTG or NCG (not even sure if that will work as well).

So what are the options? I might try and see if I can make a couple of silent mutations in the sgRNA-binding sequence so that will be different enough, or should I start looking for different sgRNA? The region I am looking at does not have many high scoring options for that, so any other nice tips / ideas?

Anoeska

[Joe Miano]

We did somethling like what you are doing w/o changing PAM in repair template.  We got 3/18 targeted pups including one where both alleles were targeted.  I think the Cas9 will be degraded and gone.  Stable genome editing will work w/o changing the PAM. 

[Ali, AM]

Anoeska,

If you cannot mutate the PAM site then try creating silent mutation within the guide sequence near to PAM. Cas9 system is sensitive to changes in the guide near PAM. I have experience with one guide that will not cut the DNA when there is one mismatch. To be sure try creating at least 3 mismatches within the guide.

Best,

Ali

On Wednesday, June 25, 2014 5:58:44 AM UTC-4, Anoeska wrote:

[Anoeska]

Thanks for the replies!

I have looked at my guide sequences and will change them so I will get mismatches (but silent mutations), see if that will work for me. 

Best,

Anoeska

Op woensdag 25 juni 2014 22:58:02 UTC+2 schreef Ali, AM:

[malz]

Hi,

I want to introduce a point mutation, however introducing silent mutation in the repair template is not desirable in my case as i am studying changes in splicing patterns. is there any alternative way of preventing the repair template from being cut by Cas9 without introducing silent mutations?

Thanks!!