Unraveling the mechanisms of PAMless DNA interrogation by SpRY-Cas9.

CRISPR-Cas9 is a powerful tool for genome editing, but the strict requirement for an NGG protospacer-adjacent motif (PAM) sequence immediately next to the DNA target limits the number of editable genes. Recently developed Cas9 variants have been engineered with relaxed PAM requirements, including SpG-Cas9 (SpG) and the nearly PAM-less SpRY-Cas9 (SpRY). However, the molecular mechanisms of how SpRY recognizes all potential PAM sequences remains unclear. Here, we combine structural and biochemical approaches to determine how SpRY interrogates DNA and recognizes target sites. Divergent PAM sequences can be accommodated through conformational flexibility within the PAM-interacting region, which facilitates tight binding to off-target DNA sequences. Nuclease activation occurs ~1000-fold slower than for Streptococcus pyogenes Cas9, enabling us to directly visualize multiple on-pathway intermediate states. Experiments with SpG position it as an intermediate enzyme between Cas9 and SpRY. Our findings shed light on the molecular mechanisms of PAMless genome editing.

Structural snapshots of R-loop formation by a type I-C CRISPR Cascade.

Type I CRISPR-Cas systems employ multi-subunit Cascade effector complexes to target foreign nucleic acids for destruction. Here, we present structures of D. vulgaris type I-C Cascade at various stages of double-stranded (ds)DNA target capture, revealing mechanisms that underpin PAM recognition and Cascade allosteric activation. We uncover an interesting mechanism of non-target strand (NTS) DNA stabilization via stacking interactions with the "belly" subunits, securing the NTS in place. This "molecular seatbelt" mechanism facilitates efficient R-loop formation and prevents dsDNA reannealing. Additionally, we provide structural insights into how two anti-CRISPR (Acr) proteins utilize distinct strategies to achieve a shared mechanism of type I-C Cascade inhibition by blocking PAM scanning. These observations form a structural basis for directional R-loop formation and reveal how different Acr proteins have converged upon common molecular mechanisms to efficiently shut down CRISPR immunity.

Constructing next-generation CRISPR-Cas tools from structural blueprints.

Clustered regularly interspaced short palindromic repeats - CRISPR-associated protein (CRISPR-Cas) systems are a critical component of the bacterial adaptive immune response. Since the discovery that they can be reengineered as programmable RNA-guided nucleases, there has been significant interest in using these systems to perform diverse and precise genetic manipulations. Here, we outline recent advances in the mechanistic understanding of CRISPR-Cas9, how these findings have been leveraged in the rational redesign of Cas9 variants with altered activities, and how these novel tools can be exploited for biotechnology and therapeutics. We also discuss the potential of the ubiquitous, yet often-overlooked, multisubunit CRISPR effector complexes for large-scale genomic deletions. Furthermore, we highlight how future structural studies will bolster these technologies.

Structural basis for mismatch surveillance by CRISPR-Cas9.

CRISPR-Cas9 as a programmable genome editing tool is hindered by off-target DNA cleavage1-4, and the underlying mechanisms by which Cas9 recognizes mismatches are poorly understood5-7. Although Cas9 variants with greater discrimination against mismatches have been designed8-10, these suffer from substantially reduced rates of on-target DNA cleavage5,11. Here we used kinetics-guided cryo-electron microscopy to determine the structure of Cas9 at different stages of mismatch cleavage. We observed a distinct, linear conformation of the guide RNA-DNA duplex formed in the presence of mismatches, which prevents Cas9 activation. Although the canonical kinked guide RNA-DNA duplex conformation facilitates DNA cleavage, we observe that substrates that contain mismatches distal to the protospacer adjacent motif are stabilized by reorganization of a loop in the RuvC domain. Mutagenesis of mismatch-stabilizing residues reduces off-target DNA cleavage but maintains rapid on-target DNA cleavage. By targeting regions that are exclusively involved in mismatch tolerance, we provide a proof of concept for the design of next-generation high-fidelity Cas9 variants.

Mesenchymal and MAPK Expression Signatures Associate with Telomerase Promoter Mutations in Multiple Cancers.

In a substantial fraction of cancers TERT promoter (TERTp) mutations drive expression of the catalytic subunit of telomerase, contributing to their proliferative immortality. We conducted a pan-cancer analysis of cell lines and find a TERTp mutation expression signature dominated by epithelial-to-mesenchymal transition and MAPK signaling. These data indicate that TERTp mutants are likely to generate distinctive tumor microenvironments and intercellular interactions. Analysis of high-throughput screening tests of 546 small molecules on cell line growth indicated that TERTp mutants displayed heightened sensitivity to specific drugs, including RAS pathway inhibitors, and we found that inhibition of MEK1 and 2, key RAS/MAPK pathway effectors, inhibited TERT mRNA expression. Consistent with an enrichment of mesenchymal states in TERTp mutants, cell lines and some patient tumors displayed low expression of the central adherens junction protein E-cadherin, and we provide evidence that its expression in these cells is regulated by MEK1/2. Several mesenchymal transcription factors displayed elevated expression in TERTp mutants including ZEB1 and 2, TWIST1 and 2, and SNAI1. Of note, the developmental transcription factor SNAI2/SLUG was conspicuously elevated in a significant majority of TERTp-mutant cell lines, and knock-down experiments suggest that it promotes TERT expression. IMPLICATIONS: Cancers harboring TERT promoter mutations are often more lethal, but the basis for this higher mortality remains unknown. Our study identifies that TERTp mutants, as a class, associate with a distinct gene and protein expression signature likely to impact their biological and clinical behavior and provide new directions for investigating treatment approaches for these cancers.