ssODN Design Question - Sense vs Antisense

[dan lichtenstein]

Dear fellow genome engineers,

I was hoping someone could explain a phenomenon of ssODN design that I am fully aware of but do not understand. When designing my ssODN templates in the past, I began with the PAM containing sequence and created silent mutations such that the PAM itself was interrupted or other mutations were introduced 5' of the PAM to decrease gRNA binding and ssODN template destruction. 

Having found a strong guide, I am now designing both antisense and sense ssODN templates with the hopes that one will more efficiently induce HDR. I understand that, if I do not to introduce modifications to the antisense PAM (ie 5' - CCN - 3'), the ssODN will be cut. However, I cannot fathom why this would be, given that the transcribed guide RNA ought to be antisense itself. 

Yours,

A confused lab tech.

[Matthew Thompson]

Cas9 does cleave ssDNA, though not as efficiently as dsDNA (source), so like you said, it's good to make the gRNA target sequence identical to the ssODN sequence so it won't match. However, after HR occurs there's a risk the newly incorporated donor template will be cleaved, since it will be dsDNA bearing the target sequence necessary for gRNA recognition.

I don't have much experience with mutating PAMs - my gRNAs overlap the SNP site I'm working with so I just called it good, but I would be worried about unkowingly creating splice variants by making "silent" mutations. Just something to watch out for.

Best,

Matt

[dan lichtenstein]

Thanks for the response! That makes a lot of sense.

New splice variants are something I worry about as well, so I try and change them the smallest amount possible. However, you probably do have some HDR cutting with only one change (unless the SNP happens to be in the PAM). The other reason I include them is for screening purposes - by introducing 1 or 2 silent mutations it is generally easy to create a new restriction site and thus screen en masse.

Best

Dan

[Joe Miano]

Best to keep changes to absolute minimum, esp with SNP modeling.  See Suppl Fig 1 in attached for a multiplex PCR genotyping assay we routinely use that works on some (though not all) SNP mice (should apply to cells as well).

[niksasi]

does anyone know if there is any advantage to transfecting both sense and antisense templates simultaneously, does that increase efficiency in any way?

Thank you this forum is aweseome!!!

On Monday, January 11, 2016 at 9:45:04 PM UTC-5, dan lichtenstein wrote:

[Matthew Thompson]

Probably not a good idea, they would almost certainly anneal to each other and you might as well try transfecting dsDNA.