3
$\begingroup$

CAS9 is the RNA-guided endonuclease that cleaves DNA as specified by the RNA sequence and is used to target viruses that infect bacteria. So why doesn't this RNA+endonuclease combo also cleave the original CRISPR spacer sequences (which also have the same DNA?). Is it the same reason that the bacterial genome is methylated (and hence protects from restriction enzymes)? Does methylation also make a difference to CAS9 as it does to restriction enzymes?

This actually was a doubt asked to Eric Lander in this lecture.

$\endgroup$

2 Answers 2

3
$\begingroup$

It is not the methylation status. The crRNA is not only complementary to the spacer sequence within the CRISPR array but also to the repeat sequence flanking that spacer. The additional base pairing of the sgRNA with the repeat prevents a nucleolytic cleavage by Cas9. In addition, the arrays typically do not contain PAM sequences.

Here you will find a nice illustration:

http://www.nature.com/nrg/journal/v11/n3/images/nrg2749-f4.jpg

$\endgroup$
0
$\begingroup$

The explanation regarding complementarity of the 5' handle is true for Type III CRISPR-Cas systems. In type II systems such as Cas9 a protospacer adjacent motif is required for self vs non-self recognition.

PS: If this question was asked to Eric Lander and he was obviously not able to answer (otherwise why is the question asked here?) then I am not surprised as to why his "Heroes of CRISPR" is so inaccurate and misrepresenting.

$\endgroup$
1
  • 1
    $\begingroup$ Please add some references to your answer. $\endgroup$ Feb 22, 2017 at 13:16

You must log in to answer this question.

Not the answer you're looking for? Browse other questions tagged .