Seminal 2014 paper showing “mismatches at dCas9 binding sites can be as high as ten”

 

This seminal 2014 paper shows the real problem of Cas9, it is meant to be non-specific – else viruses would evade it in a day

1. “mismatches at dCas9 binding sites can be as high as 10“
2. “as many as 9 of the mismatches can be consecutive in the PAM-distal region”
3. “a perfect match of 10 bases in the PAM-proximal region of the sgRNA guiding sequence is sufficient to mediate Cas9 binding to DNA.”

 

And once it binds it will cut sometimes – we just dont have the tech to observe it

Here is an example with 7 mismatches. “And with 7 mismatches (maybe more) allowed, there are 100k possible off-targets for any gRNA in most any genome.”

Fig 2 in https://www.ncbi.nlm.nih.gov/pubmed/24837660

Btw, dCas9 itself has endonuclease activity