PhD student, Max Planck Institute for Terrestrial Microbiology
A CRISPR-Cas complex that is able to perform all of its duties while lacking two relevant proteins.
As bacteria are constantly under attack by phages, there are several ways by which they fight back. CRISPR-Cas systems are one way, as these adaptive immune systems allow the cells to have a memory of past infections in the form of a CRISPR array, which helps to rapidly recognize and eliminate the invading threat. They do so through protein complexes that carry an specific RNA codified inside the CRISPR array (crRNA), and are able to scan through the DNA for their complementary viral target. Recognition starts by reading a 2 to 5 nucleotide sequence called Protospacer Adjacent Motif (PAM), which is located only on foreign genetic material and not in the host genome. This prevents the system from targeting itself, which would lead to cell death. To date, 6 types of CRISPR-Cas systems have been described, all with different sets of Cas proteins doing the job. We study the most widespread one, Type I, from which we specialize on a variant of the subtype Type I-F. Interestingly, this system´s complex lacks two of the Cas proteins described in other systems as essential for PAM recognition and target binding. Despite being one of the smallest complexes studied so far, it is able to fulfill the same roles as the bigger ones with comparable efficiency. Our work describes that it does so by having two newly characterized Cas proteins, that have features able to mimic the missing proteins. But why does this system exists on the first place when the other ones work so well? Viruses, in an attempt to by-pass the cell´s defenses, evolved Anti-CRISPR proteins, able to block the activity of several Type I and II complexes. For the first type, it has been described that the targets for this inhibitors are regions that are not conserved in the minimal Type I-F variant, and actually were exchanged by these two new Cas proteins. We hypothesize that the complex that we study underwent several modifications in time, in order to outsmart the counterattack by viruses, that way saving its host from death.
Abstract: Type I CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-Cas (CRISPR-associated) systems exist in bacterial and archaeal organisms and provide immunity against foreign DNA. The Cas protein content of the DNA interference complexes (termed Cascade) varies between different CRISPR-Cas subtypes. A minimal variant of the Type I-F system was identified in proteobacterial species including Shewanella putrefaciens CN-32. This variant lacks a large subunit (Csy1), Csy2 and Csy3 and contains two unclassified cas genes. The genome of S. putrefaciens CN-32 contains only five Cas proteins (Cas1, Cas3, Cas6f, Cas1821 and Cas1822) and a single CRISPR array with 81 spacers. RNA-Seq analyses revealed the transcription of this array and the maturation of crRNAs (CRISPR RNAs). Interference assays based on plasmid conjugation demonstrated that this CRISPR-Cas system is active in vivo and that activity is dependent on the recognition of the dinucleotide GG PAM (Protospacer Adjacent Motif) sequence and crRNA abundance. The deletion of cas1821 and cas1822 reduced the cellular crRNA pool. Recombinant Cas1821 was shown to form helical filaments bound to RNA molecules, which suggests its role as the Cascade backbone protein. A Cascade complex was isolated which contained multiple Cas1821 copies, Cas1822, Cas6f and mature crRNAs.
Pub.: 10 Sep '15, Pinned: 18 Aug '17
Abstract: Shewanella putrefaciens CN-32 contains a single Type I-Fv CRISPR-Cas system which confers adaptive immunity against bacteriophage infection. Three Cas proteins (Cas6f, Cas7fv, Cas5fv) and mature CRISPR RNAs were shown to be required for the assembly of an interference complex termed Cascade. The Cas protein-CRISPR RNA interaction sites within this complex were identified via mass spectrometry. Additional Cas proteins, commonly described as large and small subunits, that are present in all other investigated Cascade structures, were not detected. We introduced this minimal Type I system in Escherichia coli and show that it provides heterologous protection against lambda phage. The absence of a large subunit suggests that the length of the crRNA might not be fixed and recombinant Cascade complexes with drastically shortened and elongated crRNAs were engineered. Size-exclusion chromatography and small-angle X-ray scattering analyses revealed that the number of Cas7fv backbone subunits is adjusted in these shortened and extended Cascade variants. Larger Cascade complexes can still confer immunity against lambda phage infection in E. coli Minimized Type I CRISPR-Cas systems expand our understanding of the evolution of Cascade assembly and diversity. Their adjustable crRNA length opens the possibility for customizing target DNA specificity.
Pub.: 25 May '16, Pinned: 18 Aug '17
Abstract: Type I CRISPR systems feature a sequential dsDNA target searching and degradation process, by crRNA-displaying Cascade and nuclease-helicase fusion enzyme Cas3, respectively. Here we present two cryo-EM snapshots of the Thermobifida fusca type I-E Cascade: (1) unwinding 11 bp of dsDNA at the seed-sequence region to scout for sequence complementarity, and (2) further unwinding of the entire protospacer to form a full R-loop. These structures provide the much-needed temporal and spatial resolution to resolve key mechanistic steps leading to Cas3 recruitment. In the early steps, PAM recognition causes severe DNA bending, leading to spontaneous DNA unwinding to form a seed-bubble. The full R-loop formation triggers conformational changes in Cascade, licensing Cas3 to bind. The same process also generates a bulge in the non-target DNA strand, enabling its handover to Cas3 for cleavage. The combination of both negative and positive checkpoints ensures stringent yet efficient target degradation in type I CRISPR-Cas systems.
Pub.: 01 Jul '17, Pinned: 18 Aug '17
Abstract: Bacteria and archaea use CRISPR-Cas adaptive immune systems to defend themselves from infection by bacteriophages (phages). These RNA-guided nucleases are powerful weapons in the fight against foreign DNA, such as phages and plasmids, as well as a revolutionary gene editing tool. Phages are not passive bystanders in their interactions with CRISPR-Cas systems, however; recent discoveries have described phage genes that inhibit CRISPRCas function. More than 20 protein families, previously of unknown function, have been ascribed anti-CRISPR function. Here, we discuss how these CRISPR-Cas inhibitors were discovered and their modes of action elucidated. We also consider the potential impact of anti-CRISPRs on bacterial and phage evolution. Finally, we speculate about the future of this field. Expected final online publication date for the Annual Review of Virology Volume 4 is September 29, 2017. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.
Pub.: 28 Jul '17, Pinned: 18 Aug '17