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As a computer scientist, I'm interested in the ability of the Cas9 protein to function as an if-gate for DNA. It opens up so many questions for me. Right now the protein works as "if the sequence matches, cleave", but perhaps you could customize it to do "if the sequence matches, [do arbitrary action]". However, I'd feel very dumb if I got all excited about this and it tuned out "no... there are plenty of things that function as [if sequence matches], the real revolution of CRISPR/Cas9 is... [something else]". Is my fear qualified?

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There are two classes of proteins that I can think of off the bat that are DNA sequence-specific. First are restriction enzymes, which recognize a specific (usually short) sequence of DNA and cleave it, sometimes through both strands at the same spot (blunt ends) and sometimes leaving an overhang or "sticky end".

The other class is transcription factors. Instead of altering the DNA, these proteins bind to a consensus sequence and act as a gathering point for the transcription machinery to bind and begin transcribing the following gene into RNA.

What makes the CRISPR/Cas discovery unique is that the binding sequence can essentially be an arbitrary sequence, defined by the scientist, allowing for cuts just about anywhere. Transcription factors and restriction enzymes depend on the local sequence of the DNA itself, so they only bind in certain spots.

There are other sequence-specific technologies available, such as TALEN, but they are basically a combination of a site-specific transcription factor and a fairly non-specific restriction enzyme or nuclease fragment engineered into it that cuts at a specific, user-defined site, depending on which subunits are used. CRISPR is a much more powerful (and easier to implement in vivo) technology, though, so while TALEN still has a place in the molecular biology toolbox, CRISPR is rapidly eclipsing it.

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    $\begingroup$ Just a minor thing but the TALEN class of nucleases are actually transcription factors with a FokI nuclease domain attached to it. They resemble the old zinc finger nucleases in that they are DNA recognition proteins with a nuclease attached to it. CRISPR is unique in that it is a nuclease in its original form. $\endgroup$ – mimat Oct 16 '16 at 15:50
  • $\begingroup$ @mimat yes, you're right. I wrote that last section quickly, based off my reading on TALENs a little while back. I didn't remember that they actually have TF activities as well. I reworded that section a bit. $\endgroup$ – MattDMo Oct 16 '16 at 15:55
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OK, if you want analogy, you can think of Cas9 as comparing device: if sgRNA (guide RNA) matches DNA, then cleave. You can pick this up from Wiki article or publications on CRISPR/Cas9

Read about DNA restriction enzymes. If in case of Cas9 template is provided by sgRNA (guide RNA), in case of restriction enzymes template is built-in this enzyme.

Cool thing about Cas9/CRISPR is that you can engineer guide RNA to match 20 bases of DNA for high specificity.

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