Cryo-EM Structures Reveal Mechanism and Inhibition of DNA Targeting by a CRISPR-Cas Surveillance Complex
Electron-microscopy images reveal how a CRISPR system marks specific DNA sequences for destruction. Microbes use CRISPR as a defense system to fend off viruses and other invaders, and geneticists have harnessed it to alter DNA sequences in a process called gene editing. We used cryo-electron microscopy to watch a CRISPR system from the bacterium Pseudomonas aeruginosa as it bound to DNA. We found that this process causes large structural changes in an enzyme complex called Csy — which recognizes the target DNA — and the fragment of ‘guide’ RNA that steers Csy to its target. These changes could be important for allowing other enzymes to cut the targeted DNA.
– Cryo-EM structures of Csy surveillance complex with and without bound dsDNA.
– dsDNA target binding induces change in pitch of Csy complex.
– Visualization of PAM recognition and spacer:protospacer heteroduplex formation.
– Binding site definition of anti-CRISPR inhibitors that block dsDNA binding.
| Abstract | Prokaryotic cells possess CRISPR-mediated adaptive immune systems that protect them from foreign genetic elements, such as invading viruses. A central element of this immune system is an RNA-guided surveillance complex capable of targeting non-self DNA or RNA for degradation in a sequence- and site-specific manner analogous to RNA interference. Although the complexes display considerable diversity in their composition and architecture, many basic mechanisms underlying target recognition and cleavage are highly conserved. Using cryoelectron microscopy (cryo-EM), we show that the binding of target double-stranded DNA (dsDNA) to a type I-F CRISPR system yersinia (Csy) surveillance complex leads to large quaternary and tertiary structural changes in the complex that are likely necessary in the pathway leading to target dsDNA degradation by a trans-acting helicase-nuclease. Comparison of the structure of the surveillance complex before and after dsDNA binding, or in complex with three virally encoded anti-CRISPR suppressors that inhibit dsDNA binding, reveals mechanistic details underlying target recognition and inhibition. |
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