Science1 publisher2 min readPublished
Structure search for CRISPR's ancestor turns up a viral protein that opens DNA with a triplex
Two Science papers name the protein VIPR and show it silencing a reporter gene and blocking phage in E. coli, guided by an RNA that reads two DNA letters and skips the third, a targeting rule anyone borrowing it will have to design around.
The Scientist · Science desk
What happened
- Two studies published in Science describe an RNA-guided system that originated in viruses themselves and that the authors argue was a precursor to bacterial CRISPR.
- A structure-based search for relatives of the RAMP proteins that define class 1 CRISPR turned up an RNA-binding protein the team named VIPR, for viral interference programmable repeat.
- In E. coli, VIPR bound DNA, could be reprogrammed to silence a fluorescent reporter gene, and protected the bacteria against an invading phage.
- Its guide RNA reads two DNA letters, skips one and reads the next two, where a CRISPR guide matches its target in a single continuous stretch.
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Why it matters
- capability Protein-driven triplex formation is a second physical route to opening a DNA duplex under RNA guidance, so a protein engineer is no longer restricted to the R-loop chemistry that known RNA-guided systems use.
- constraint What has been shown is binding and silencing in bacteria, so a group wanting a cut or a base change inherits a scaffold and has to supply the effector domain itself.
- decision Targeting rules cannot be carried over from CRISPR guide design, because a discontinuous guide reads fewer bases than the stretch of DNA it covers.
The part of this work with experiments attached is the mechanics. Known RNA-guided systems open a DNA duplex by pairing: the guide RNA invades the helix, displaces one strand and leaves an R-loop. VIPR opens it another way. "Despite architectural parallels to CRISPR-Cas, VIPR systems unwind DNA by a mechanism distinct from known RNA-guided systems, which rely on base pairing-driven R-loop formation," the study authors wrote [15]. "Rather than invading the duplex through RNA strand exchange, VIPR systems use protein-driven triplex formation to unwind the DNA and enable RNA-DNA pairing" [16]. In the structural images the protein wraps the guide RNA and both DNA strands into an unusual three-stranded shape, which is how it pries the duplex open [14].
The skip-one code fits that geometry. A guide that reads two bases, skips one and reads two more is checking two of every three positions it spans, so twenty matched bases occupy about thirty bases of target [11][19]. Whether that costs specificity in a genome much larger than a phage's is not a question a reporter-gene assay in E. coli settles [13]. The guides themselves are built from repeated units, with conserved segments the protein holds in place and variable segments that set the DNA target [12].
On the evidence in these papers, VIPR is a programmable DNA-binding module with silencing activity in bacteria [13]. A nuclease, a base editor or a methyltransferase would be somebody's fusion partner. The researchers call VIPR systems the most minimal and potentially versatile platforms for RNA-guided DNA recognition encountered so far [17], and they name genome editing, DNA locus imaging and epigenetic modification as uses for synthetic VIPR fusions [18]. The experiments described are all in E. coli [13].
RAMPs are why anyone went looking. "RAMPs are among the most conserved and ancient features of CRISPR-Cas, proposed to date back to the last universal common ancestor," the authors wrote [5], and their origins had been especially unclear [20]. The team searched by structure, and turned up VIPR in both viral and bacterial genomes, alongside the small noncoding vrRNA that guides it [6][7]. Because natural VIPR targets fell so often inside rival bacteriophages, the team proposes the system began as a way for viruses to inactivate each other while competing inside an invaded bacterium [8], with bacteria later co-opting it for their own defense and CRISPR emerging from that [9]. The account published by phys.org describes the proposed link as strongly supported but says there is currently no direct evidence, and the team says identifying intermediate systems between VIPR and class 1 CRISPR would further support the model [10].
What to watch
- Publication of intermediate systems sitting between VIPR and class 1 CRISPR, the evidence the team itself named as the test of its evolutionary model.
- Any VIPR fusion carrying a nuclease or a methyltransferase working outside E. coli, reported with off-target measurements.
- Whether the skip-one targeting rule holds up against a large genome rather than a reporter gene in bacteria.