Cas13d, the type VI-D CRISPR-Cas effector, is an RNA-guided ribonuclease that has been repurposed to edit RNA in a programmable manner. Here we report the detailed structural and functional analysis of the uncultured Ruminococcus sp . Cas13d (UrCas13d)-crRNA complex. Two hydrated Mg 2+ ions aid in stabilizing the conformation of the crRNA repeat region. Sequestration of divalent metal ions does not alter pre-crRNA processing, but abolishes target cleavage by UrCas13d. Notably, the pre-crRNA processing is executed by the HEPN-2 domain. Furthermore, both the structure and sequence of the nucleotides U(-8)-C(-1) within the repeat region are indispensable for target cleavage, and are specifically recognized by UrCas13d. Moreover, correct base pairings within two separate spacer regions (an internal and a 3′-end region) are essential for target cleavage. These findings provide a framework for the development of Cas13d into a tool for a wide range of applications.

Dear Editor, CRISPR-Cas (clustered regularly interspaced short palindromic repeats and CRISPR-associated proteins) systems are RNA-guided adaptive immune systems in prokaryotes. 1,2 Class 2 CRISPR-Cas systems (including type II, V, and VI) involve large single effector proteins in complex with crRNA for interference. 3,4 The type II and V effectors, such as Cas9 and Cas12a, have been engineered into powerful tools for genome editing. The type VI system encompasses RNA-guided RNases. Its effectors Cas13a, Cas13b and Cas13d are capable of both precursor CRISPR RNA (pre-crRNA) processing and target RNA cleavage, which protect the host from phage attacks. 5-7 Once bound to a target RNA, they are activated, switching on a non-specific RNase activity. Moreover, they have been utilized to target and edit RNA as programmable RNAbinding modules. 6,[8][9][10][11][12] Although related to Cas13a and Cas13d, Cas13b possesses many distinctive features. These include the lack of significant sequence similarity with Cas13a and Cas13d, disparate crRNA repeat region, double-sided protospacer flanking sequence (PFS)-dependent target RNA cleavage. [5][6][7][8]13 To investigate how Cas13b processes pre-crRNA, recognizes crRNA and settles the spacer nucleotides for target recognition, we solved the crystal structure of Bergeyella zoohelcum Cas13b (BzCas13b) in complex with its crRNA at 2.79 Å resolution (Supplementary information, Table S1). The binary complex was obtained by the SeMet-derived BzCas13b R1177A mutant co-expressed with CRISPR template in vivo. The architecture of BzCas13b assumes a triangular domain distribution around the central L-shaped crRNA ( Fig. 1a-e; Supplementary information, Movie S1). In the binary complex, Helical-1, HEPN-1 and HEPN-2 domains together form one side of the triangular structure. Helical-1 domain comprises six α-helices connected with random loops (Supplementary information, Fig. S1). The second side of the triangle is formed by RRI-1 (the repeat region interacting domain-1), RRI-2 domains and the linker region. RRI-1 domain can be subdivided into two separate motifs (RRI-1 I and II) that stack onto each other. Both motifs contain a short twostranded, antiparallel β-sheet flanked by five α-helices. RRI-2 domain includes a long central two-stranded, antiparallel β-sheet flanked by two α-helices, and a short central two-stranded, antiparallel β-sheet flanked by three α-helices. The linker region consists of random loops that connect two short α-helices, which shows multiple interactions with RRI-2 domain. Helical-2 domain is composed of nine α-helices and its rather long helix-23 extends in parallel with crRNA, thereby forming the third side of the triangle. Helix-8 of Helical-1 domain and helix-23 of Helical-2 domain protrude out of the complex in a crab claw-like manner to clamp the spacer region of crRNA (Supplementary information, Fig. S1). In addition, HEPN-1 domain bridges Helical-1 and Helical-2 domains.A mature 52-nt crRNA, originated from a co-expressed CRISPR encoding sequence and bein...

Using bacteriophage-derived endolysins as an alternative strategy for fighting drug-resistant bacteria has recently been garnering renewed interest. However, their application is still hindered by their narrow spectra of activity. In our previous work, we demonstrated that the endolysin LysIME-EF1 possesses efficient bactericidal activity against multiple strains of Enterococcus faecalis (E. faecalis). Herein, we observed an 8 kDa fragment and hypothesized that it contributes to LysIME-EF1 lytic activity. To examine our hypothesis, we determined the structure of LysIME-EF1 at 1.75 Å resolution. LysIME-EF1 exhibits a unique architecture in which one full-length LysIME-EF1 forms a tetramer with three additional Cterminal cell-wall binding domains (CBDs) that correspond to the abovementioned 8 kDa fragment. Furthermore, we identified an internal ribosomal binding site (RBS) and alternative start codon within LysIME-EF1 gene, which are demonstrated to be responsible for the translation of the truncated CBD. To elucidate the molecular mechanism for the lytic activity of LysIME-EF1, we combined mutagenesis, lytic activity assays and in vivo animal infection experiments. The results confirmed that the additional LysIME-EF1 CBDs are important for LysIME-EF1 architecture and its lytic activity. To our knowledge, this is the first determined structure of multimeric endolysin encoded by a single gene in E. faecalis phages. As such, it may provide valuable insights into designing potent endolysins against the opportunistic pathogen E. faecalis.

Mangrove swamp is one of the world’s richest and most productive marine ecosystems. This ecosystem also has a great ecological importance, but is highly susceptible to anthropogenic disturbances. The balance of mangrove ecosystem depends largely on the microbial communities in mangrove sediments. Thus, understanding how the mangrove microbial communities respond to spatial differences is essential for more accurate assessment of mangrove ecosystem health. To this end, we performed the first medium-distance (150 km) research on the biogeographic distribution of mangrove microbial communities. The hypervariable regions of 16S rRNA gene was sequenced by Illumina to compare the microbial communities in mangrove sediments collected from six locations (i.e. Zhenzhu harbor, Yuzhouping, Maowei Sea, Qinzhou harbor, Beihai city and Shankou) along the coastline of Beibu Gulf in Guangxi province, China. Collectively, Proteobacteria , Bacteroidetes , Chloroflexi , Actinobacteria , Parvarchaeota , Acidobacteria and Cyanobacteria were the predominant phyla in the mangrove sediments of this area. At genus level, the heat map of microbial communities reflected similarities between study sites and was in agreement with their biogeographic characteristics. Interestingly, the genera Desulfococcus , Arcobacter , Nitrosopumilus and Sulfurimonas showed differences in abundance between study sites. Furthermore, the principal component analysis (PCA) and unweighted UniFrac cluster tree of beta diversity were used to study the biogeographic diversity of the microbial communities. Relatively broader variation of microbial communities was found in Beihai city and Qinzhou harbour, suggesting that environmental condition and historical events may play an important role in shaping the bacterial communities as well. This is the first report on medium-distance range distribution of bacteria in the mangrove swamp ecosystem. Our data is valuable for monitoring and evaluation of the impact of human activity on mangrove habitats from the perspective of microbiome.

Proteasomes are highly abundant and conserved protease complexes that eliminate unwanted proteins in the cells. As a single-chain ATP-independent nuclear proteasome activator, proteasome activator 200 (PA200) associates with 20S core particle to form proteasome complex that catalyzes polyubiquitin-independent degradation of acetylated histones, thus playing a pivotal role in DNA repair and spermatogenesis. Here, we present cryo-electron microscopy (cryo-EM