CRISPR and the structural perspective

Image: RNA and DNA in Cascade complex. 3D coordinates are taken from the Protein Data Bank (PDB 4QYZ).

Last term, I was doing teaching assistant duty for undergraduate Structural Biology computer lab course. I had some freedom in deciding the biomolecules that they were going to explore, so I decided why not let them explore the structure of CRISPR/Cas system?

By now, you might have heard of the gene editing tool CRISPR/Cas system (usually referred to as just “CRISPR” in the media). Even if you don’t use it in your research (I don’t), I think it is useful to have some basic understanding of it, since it has entered the public consciousness, and in my opinion it is the duty of the scientist to communicate and translate the jargons to the layman.

For my part, in the attempts to understand and to be understood, I have asked a colleague who used CRISPR herself and also tried to explain it to a friend (engineer, not scientist). The Wikipedia page proved to be comprehensive, but difficult to parse. I have found this video from Wired useful:

So, back to my students. If after all that reading or watching, you are left scratching your head and asking: “But how does CRISPR really look like at the molecular level?” then you have the talent to become a wiza-, I mean, a structural biologist. I want my students to see how a structural perspective helps us understand function. That previous sentence is basically one of the core tenets of structural biology. One manifestation of this principle is the lock and key model: there is a particular shape of an enzyme which fits a particular shape of a ligand, in order to perform its functions. Rational drug design also departs from the same reasoning — that a drug molecule binds to a target protein and causes some biological response, and by knowing the shape of the target protein, one can design a better-binding drug.

crispr

Structure of DNA-bound Cascade (PDB 4QYZ). RNA guide in blue; Single-stranded DNA target in red.

Now here are some questions that I posed to my students based on the structure of a particular CRISPR/Cas system shown above. Can you get the answers right? To view the answers, highlight the blank space.

  1. Does the DNA-RNA form a secondary structure? How is its structure related to the function of the complex?
    • It does not form a regular double helix, but a ladder-like unwound helix, with complementary Watson-Crick base pairing. Looser binding might enable better scanning of target sequence; regular kink every 6th basepair suggests conformational proofreading mechanism (see paper).
  2. Which is more exposed to the solvent, DNA or RNA? Why?
    • DNA. RNA is the guide, and its phosphate-sugar backbone is bound to the Cas proteins; DNA is the target, so its phosphate-sugar backbone is exposed to the solvent.

The paper

Mulepati, Sabin, Annie Héroux, and Scott Bailey. “Crystal structure of a CRISPR RNA–guided surveillance complex bound to a ssDNA target.” Science 345.6203 (2014): 1479-1484. (link)

Further reading

Other CRISPR structures in the Protein Data Bank.
Wired: Crispr’s Next Big Debate: How Messy Is Too Messy?
Review in Nature Microbiology: A decade of discovery: CRISPR functions and applications

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