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Canonical CRISPR-Cas systems provide adaptive immunity against mobile genetic elements.sup.1. However, type I-F, I-B and V-K systems have been adopted by Tn7-like transposons to direct RNA-guided transposon insertion.sup.2-7. Type V-K CRISPR-associated transposons rely on the pseudonuclease Cas12k, the transposase TnsB, the AAA+ ATPase TnsC and the zinc-finger protein TniQ.sup.7, but the molecular mechanism of RNA-directed DNA transposition has remained elusive. Here we report cryo-electron microscopic structures of a Cas12k-guide RNA-target DNA complex and a DNA-bound, polymeric TnsC filament from the CRISPR-associated transposon system of the photosynthetic cyanobacterium Scytonema hofmanni. The Cas12k complex structure reveals an intricate guide RNA architecture and critical interactions mediating RNA-guided target DNA recognition. TnsC helical filament assembly is ATP-dependent and accompanied by structural remodelling of the bound DNA duplex. In vivo transposition assays corroborate key features of the structures, and biochemical experiments show that TniQ restricts TnsC polymerization, while TnsB interacts directly with TnsC filaments to trigger their disassembly upon ATP hydrolysis. Together, these results suggest that RNA-directed target selection by Cas12k primes TnsC polymerization and DNA remodelling, generating a recruitment platform for TnsB to catalyse site-specific transposon insertion. Insights from this work will inform the development of CRISPR-associated transposons as programmable site-specific gene insertion tools. Structural studies on Scytonema hofmanni CRISPR-associated transposon protein complexes indicate a mechanism for RNA-guided DNA transposition involving Cas12k, TnsC and TnsB., Author(s): Irma Querques [sup.1] , Michael Schmitz [sup.1] , Seraina Oberli [sup.1] , Christelle Chanez [sup.1] , Martin Jinek [sup.1] Author Affiliations: (1) Department of Biochemistry, University of Zurich, Zurich, [...]

In many prokaryotes, type III clustered regularly interspaced short palindromic repeat (CRISPR)CRISPR-associated (Cas) systems detect and degrade invasive genetic elements by an RNA-guided, RNA-targeting multisubunit interference complex. The CRISPR-associated protein Csm6 additionally contributes to interference by functioning as a standalone RNase that degrades invader RNA transcripts, but the mechanism linking invader sensing to Csm6 activity is not understood. Here we show that Csm6 proteins are activated through a second messenger generated by the type III interference complex. Upon target RNA binding by the interference complex, its Cas10 subunit converts ATP into a cyclic oligoadenylate product, which allosterically activates Csm6 by binding to its CRISPR-associated Rossmann fold (CARF) domain. CARF domain mutations that abolish allosteric activation inhibit Csm6 activity in vivo, and mutations in the Cas10 Palm domain phenocopy loss of Csm6. Together, these results point to an unprecedented mechanism for regulation of CRISPR interference that bears striking conceptual similarity to oligoadenylate signalling in mammalian innate immunity., Author(s): Ole Niewoehner [1]; Carmela Garcia-Doval [1]; Jakob T. Rostl [2]; Christian Berk [3]; Frank Schwede [4]; Laurent Bigler [5]; Jonathan Hall [3]; Luciano A. Marraffini [2]; Martin Jinek (corresponding [...]

The CRISPR-associated protein Cas9 is an RNA-guided endonuclease that cleaves double-stranded DNA bearing sequences complementary to a 20-nucleotide segment in the guide RNA (1,2). Cas9 has emerged as a versatile molecular tool for genome editing and gene expression control (3). RNA-guided DNA recognition and cleavage strictly require the presence of a protospacer adjacent motif (PAM) in the target DNA (1,4-6). Here we report a crystal structure of Streptococcus pyogenes Cas9 in complex with a single-molecule guide RNA and a target DNA containing a canonical 5 '-NGG-3' PAM. The structure reveals that the PAM motif resides in a base-paired DNA duplex. The non-complementary strand GG dinucleotide is read out via major-groove interactions with conserved arginine residues from the carboxy-terminal domain of Cas9. Interactions with the minor groove of the PAM duplex and the phosphodiester group at the +1 position in the target DNA strand contribute to local strand separation immediately upstream of the PAM. These observations suggest a mechanism for PAM-dependent target DNA melting and RNA-DNA hybrid formation. Furthermore, this study establishes a framework for the rational engineering of Cas9 enzymes with novel PAM specificities. ED Fig. 3b, 7a,c,d Fig. 2d Fig. 3d, In type IICRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated) systems, the endonuclease Cas9 associates with a dual-RNA guide structure consisting of a CRISPR RNA (crRNA) and a trans-activating CRISPR [...]

The clustered regularly interspaced short palindromic repeats (CRISPR)-associated enzyme Cas9 is an RNA-guided endonuclease that uses RNA-DNA base-pairing to target foreign DNA in bacteria. Cas9-guide RNA complexes are also effective genome engineering agents in animals and plants. Here we use single-molecule and bulk biochemical experiments to determine how Cas9-RNA interrogates DNA to find specific cleavage sites. We show that both binding and cleavage of DNA by Cas9-RNA require recognition of a short trinucleotide protospacer adjacent motif (PAM). Non-target DNA binding affinity scales with PAM density, and sequences fully complementary to the guide RNA but lacking a nearby PAM are ignored by Cas9-RNA. Competition assays provide evidence that DNA strand separation and RNA-DNA hetero-duplex formation initiate at the PAM and proceed directionally towards the distal end of the target sequence. Furthermore, PAM interactions trigger Cas9 catalytic activity. These results reveal how Cas9 uses PAM recognition to quickly identify potential target sites while scanning large DNA molecules, and to regulate scission of double-stranded DNA., RNA-mediated adaptive immune systems in bacteria and archaea rely on CRISPRs and CRISPR-associated (Cas) proteins to provide protection from invading viruses and plasmids (1). Bacteria harbouring CRISPR-Cas loci respond to [...]

Transfer RNAs are among the most ubiquitous molecules in cells, central to decoding information from messenger RNAs on translating ribosomes. In eukaryotic cells, tRNAs are actively transported from their site of synthesis in the nucleus to their site of function in the cytosol. This is mediated by a dedicated nucleo-cytoplasmic transport factor of the karyopherin-β family (Xpot, also known as Lost in Soccharomyces cerevisiae). Here we report the 3.2 Å resolution structure of Schizosoccharomyces pornbe Xpot in complex with tRNA and RanGTP, and the 3.1 Å structure of unbound Xpot, revealing both nuclear and cytosolic snapshots of this transport factor. Xpot undergoes a large conformational change on binding cargo, wrapping around the tRNA and, in particular, binding to the tRNA 5' and 3' ends. The binding mode explains how Xpot can recognize all mature tRNAs in the cell and yet distinguish them from those that have not been properly processed, thus coupling tRNA export to quality control., During interphase, tRNAs relocate from the sites of transcription in the nucleus to the sites of translation in the cytosol (1,2). There they decode the genetic information in mRNAs by [...]

In eukaryotes, small non-coding RNAs regulate gene expression, helping to control cellular metabolism, growth and differentiation, to maintain genome integrity, and to combat viruses and mobile genetic elements. These pathways involve two specialized ribonucleases that control the production and function of small regulatory RNAs. The enzyme Dicer cleaves double-stranded RNA precursors, generating short interfering RNAs and microRNAs in the cytoplasm. These small RNAs are transferred to Argonaute proteins, which guide the sequence-specific silencing of messenger RNAs that contain complementary sequences by either enzymatically cleaving the mRNA or repressing its translation. The molecular structures of Dicer and the Argonaute proteins, free and bound to small RNAs, have offered exciting insights into the molecular mechanisms that are central to RNA silencing pathways., The discovery that RNA interference (RNAi) and related small-RNA-mediated pathways have central roles in the silencing of gene expression in eukaryotic cells has profoundly altered the understanding of gene regulation. [...]

Cas9 is a CRISPR-associated endonuclease capable of RNA-guided, site-specific DNA cleavage 1-3 . The programmable activity of Cas9 has been widely utilized for genome editing applications 4-6 , yet its precise mechanisms of target DNA binding and off-target discrimination remain incompletely understood. Here we report a series of cryo-electron microscopy structures of Streptococcus pyogenes Cas9 capturing the directional process of target DNA hybridization. In the early phase of R-loop formation, the Cas9 REC2 and REC3 domains form a positively charged cleft that accommodates the distal end of the target DNA duplex. Guide-target hybridization past the seed region induces rearrangements of the REC2 and REC3 domains and relocation of the HNH nuclease domain to assume a catalytically incompetent checkpoint conformation. Completion of the guide-target heteroduplex triggers conformational activation of the HNH nuclease domain, enabled by distortion of the guide-target heteroduplex, and complementary REC2 and REC3 domain rearrangements. Together, these results establish a structural framework for target DNA-dependent activation of Cas9 that sheds light on its conformational checkpoint mechanism and may facilitate the development of novel Cas9 variants and guide RNA designs with enhanced specificity and activity., (© 2022. The Author(s).)