Solving a targeting puzzle: Type III-B DNA cleavage
Type III CRISPR systems are unusual in part because they have multiple mechanisms for cleaving nucleic acids, and target both RNA and DNA, but the latter only in a transcription-dependent matter. A 2016 paper from the Bailey lab in Genes and Development filled in key pieces of the puzzle about Type III systems, helping to clarify two major questions: What are the specific RNA and DNA targets of Type III complex? And what mechanisms control and coordinate the RNA and DNA cleavage activities?
Around the same time, papers from two other labs showed similar results from different species: the Terns lab published a paper in the same issue of Genes and Development looking at the Pyrococcus furiosus Cmr system, and the Siksnys lab published a paper in Molecular Cell about the Streptococcus thermophilus Type III-A system.
The Bailey lab’s work was led by PhD student Michael Estrella, with support from ScM student Fang-Ting Kuo, and focused on Thermotoga maritima’s Type III-B system, which uses a Cmr CRISPR complex.
At the time, research had shown that Type III-B complexes targeted RNA, and DNA in a transcription-dependent manner. But only the RNA cleavage activity had been demonstrated in reconstituted systems in vitro, where biochemical assays could be used to probe the mechanisms.
To look closer, Michael started by purifying T. maratima Cmr proteins and combining them with crRNA to form functional complexes that could function in vitro.
He first tested the substrate specificity by incubating the complex with a single substrate: either complementary ssRNA, noncomplementary ssRNA, complementary ssDNA or dsDNA. As with previous in vitro reports, the complementary ssRNA was cleaved while the other substrates remained intact.
Michael and his colleagues reasoned that the presence of ssRNA was needed to activate the DNA cleavage, and incubated the Cmr complex with ssRNA and DNA targets together rather than individually. They found that they found that ssDNA, but not dsDNA, was weakly cut, but only when ssRNA that was complementary to the crRNA was included. Unlike the ssRNA, the ssDNA was cleaved regardless of sequence.
They also tested targeting of dsDNA with short sections of mis-matches, which form “bubbles,” regions of ssDNA where the bases couldn’t pair up. The bubble regions could be cleaved by the Cmr complex, even when they were only two bases long.
So, the complex targets ssRNA that matches the crRNA, and ssDNA (even if just a short region within dsDNA), but only in the presence of that ssRNA. But in addition to identifying the RNA and DNA targets, Michael also identified a critical timing aspect – the ssDNA wasn’t cleaved if it was added 10 minutes after the ssRNA target, rather than at the same time.
This fit with something else that Michael had discovered about the ssRNA cleavage mechanism. Like in other Type III systems, the ssRNA was cleaved in a specific pattern, with multiple cuts six bases apart from each other. Michael took it a step further to determine the timing of the cuts, and showed they were made sequentially, taking about 5 minutes to complete under the conditions he used.
So, waiting 10 minutes to add the ssDNA gave the complex time to finish cutting the ssRNA. This suggested that while ssDNA nuclease activity required ssRNA binding, it was inhibited again once the ssRNA was cleaved and released. In fact, under conditions where the Cmr complex could bind but not cut ssRNA, they saw even higher levels of ssDNA cleavage.
This suggested a mechanism for controlling of the ssDNA activity, with timing and location elements: the ssDNA cleavage is limited to regions of ssDNA near to the complex in the short period of time after the complex binds the ssRNA target but before it finishes cutting it. These limits may be especially important for the ssDNA rather than ssRNA targeting, because the ssDNA cleavage is not limited to a particular sequence that matches the crRNA, giving it the potential to cleave many unintended targets.
These results helped to resolve an open question about how the Type III complexes identified the transcriptionally active DNA target. At the time, there were two proposed models, where the complex targeted and crRNA bound to either the RNA transcript or the non-template strand of the DNA where it is exposed in the transcription bubble.
Michael’s work firmly supported the model where the complex binds to the ssRNA, and further indicated that that binding activates the ssDNA cleavage. Michael and Scott proposed that the Cmr complex binding to an RNA transcript tethers it near the phage DNA during the short time the ssDNA activity is active before ssRNA cleavage is complete. While the model in this paper, and others, suggests that the complex may cleave the exposed non-template DNA strand of the transcription bubble, the specific target of the ssDNA activity in vivo remains unclear.
To find out more about the research, including experiments that revealed more details of the ssRNA and ssDNA cleavage mechanisms, check out the paper.
The work was funded by the NIH’s National Institutes of General Medical Sciences and a Ruth L. Kirschstein National Institutes of Health F31 fellowship to Michael Estrella.
Want to learn more about the work this paper builds on? Check out these papers:
Type III system cleavage of transcriptionally active DNA
Deng L, Garrett RA, Shah SA, Peng X, She Q. A novel interference mechanism by a type IIIB CRISPR-Cmr module in Sulfolobus. Mol Microbiol, 2013.
Goldberg GW, Jiang W, Bikard D, Marraffini LA. Conditional tolerance of temperate phages via transcription-dependent CRISPR-Cas targeting. Nature, 2014.
Samai P, Pyenson N, Jiang W, Goldberg GW, Hatoum-Aslan A, Marraffini LA. Co-transcriptional DNA and RNA Cleavage during Type III CRISPR-Cas Immunity. Cell, 2015.
Type III-B Cmr systems
Hale CR, Zhao P, Olson S, Duff MO, Graveley BR, Wells L, Terns RM, Terns MP. RNA-guided RNA cleavage by a CRISPR RNA-Cas protein complex. Cell, 2009.
Hale CR, Majumdar S, Elmore J, Pfister N, Compton M, Olson S, Resch AM, Glover CV 3rd, Graveley BR, Terns RM, Terns MP. Essential features and rational design of CRISPR RNAs that function with the Cas RAMP module complex to cleave RNAs. Mol Cell, 2012.
Staals RHJ, Agari Y, Maki-Yonekura S, Zhu Y, Taylor DW, van Duijn E, Barendregt A, Vlot M, Koehorst JJ, Sakamoto K, Masuda A, Dohmae N, Schaap PJ, Doudna JA, Heck AJR, Yonekura K, van der Oost J, Shinkai A. Structure and activity of the RNA-targeting Type III-B CRISPR-Cas complex of Thermus thermophilus. Mol Cell, 2013.
Zebec Z, Manica A, Zhang J, White MF, Schleper C. CRISPR-mediated targeted mRNA degradation in the archaeon Sulfolobus solfataricus. Nucleic Acids Res, 2014.
Zhang J, Rouillon C, Kerou M, Reeks J, Brugger K, Graham S, Reimann J, Cannone G, Liu H, Albers SV, Naismith JH, Spagnolo L, White MF. Structure and mechanism of the CMR complex for CRISPR-mediated antiviral immunity. Mol Cell, 2012.
Characterization of Type III-A system Csm complexes (structurally similar to Cmr complexes)
Staals RH, Zhu Y, Taylor DW, Kornfeld JE, Sharma K, Barendregt A, Koehorst JJ, Vlot M, Neupane N, Varossieau K, Sakamoto K, Suzuki T, Dohmae N, Yokoyama S, Schaap PJ, Urlaub H, Heck AJ, Nogales E, Doudna JA, Shinkai A, van der Oost J. RNA targeting by the type III-A CRISPR-Cas Csm complex of Thermus thermophilus. Mol Cell, 2014.
Rouillon C, Zhou M, Zhang J, Politis A, Beilsten-Edmands V, Cannone G, Graham S, Robinson CV, Spagnolo L, White MF. Structure of the CRISPR interference complex CSM reveals key similarities with cascade. Mol Cell, 2013.
Tamulaitis G, Kazlauskiene M, Manakova E, Venclovas Č, Nwokeoji AO, Dickman MJ, Horvath P, Siksnys V. Programmable RNA shredding by the type III-A CRISPR-Cas system of Streptococcus thermophilus. Mol Cell, 2014.
Related papers
Other papers published at a similar time with similar results from different Type III systems
Elmore JR, Sheppard NF, Ramia N, Deighan T, Li H, Terns RM, Terns MP. Bipartite recognition of target RNAs activates DNA cleavage by the Type III-B CRISPR-Cas system. Genes Dev, 2016.
Kazlauskiene M, Tamulaitis G, Kostiuk G, Venclovas Č, Siksnys V. Spatiotemporal Control of Type III-A CRISPR-Cas Immunity: Coupling DNA Degradation with the Target RNA Recognition. Mol Cell, 2016.