The Cas9 CRISPR target: diagram

The CRISPR target as recognized by Cas9 protein + guide RNA.


  1. I'm slightly confused. According to this illustration it seems that "protospacer" is defined as the sequence identical to the crRNA sequence. Other sources state the contrary. For example, this: "For clarity, we use the definition of the protospacer as the DNA strand complementary to the crRNA, and PAMs are denoted 5'-3' on the protospacer DNA." ( Could you clarify what is the current consensus on this? Thank you :)

  2. Thanks for your question: here's my take on it. The short version of my answer is that technically, the protospacer refers to a double-stranded region of the genomic DNA in a bacterial CRISPR-encoding region or in a CRISPR cleavage target; therefore it's not clear that it "should" refer to either strand preferentially. And in most of the current literature a protospacer is used to refer to a double-stranded genomic sequence, not a specific strand.

    I admit that my diagram kinda looks like I am referring to the top strand as the protospacer and I don't have much literature basis for this. But, I guess when I think "protospacer" I automatically think of this strand because (1) for *Cas9* the PAM (protospacer-adjacent motif) is now always used in reference to the top strand, and (2) the cRNA sequence is the same as this strand. For Cas9 the PAM really sort of is the top strand since it’s now know that the protein actually contacts the guanine nucleotides (Anders et al Nature 514(751) pp569-573, 2014). However the PAM sequence only exists at the double-stranded DNA target – it doesn’t pair with the cRNA - and so the PAM never really is single-stranded.

    Note that in your cited reference (Biswas et al, RNA biology 2013) the authors had to state that they chose which strand to call the protospacer, which means it was not clearly defined in the literature prior to that. In any event I do not believe that the Biswas paper has generated a common agreement that the term refers to the complementary strand.