Prevent Cas9 from cleaving the target sequence in ssODN experiment

I wonder if I mutate several base pairs of single strand DNA oligo in PAM and upstream from the Cas9 cleavage site at the same time.

Here is the original sequence and the desired changes. TGG is PAM sequence.

 _  AGGTGCGC GTGCCAGTC GCG_ ** _TGG  _ (original)**

-> AGGTGCGC AAA CCAGTC GCG AAA (desired change)

**
**

These Cas9 cleavage preventing mutations are the point mutations that I want to make and I do not need no other changes such as insertion or deletion.

Please let me know if this strategy works.


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Sapphire Griffinover 4 years ago

That's correct. You don't need to make any other changes to block Cas9.

Coral Fenrirover 4 years ago

Does that mean both of the AAA changes will integrate successfully?

Terracotta Harpyover 4 years ago

Whether both sets of mutations get synthesized during repair and integrated into the host genome depends on the extent of resection following DSB. Generally, there is sufficient resection of the DSB to facilitate single strand template repair (SSTR). The further those AAAs are from the DSB, the less likely they get used during the SSTR process.
You should be fine...though I have experienced one instance such as yours where the PAM mutations were present but the upstream mutations were not.

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