Cas9 interrogates DNA in discrete steps modulated by mismatches and supercoiling.

The CRISPR-Cas9 nuclease has been widely repurposed as a molecular and cell biology tool for its ability to programmably target and cleave DNA. Cas9 recognizes its target site by unwinding the DNA double helix and hybridizing a 20-nucleotide section of its associated guide RNA to one DNA strand, forming an R-loop structure. A dynamic and mechanical description of R-loop formation is needed to understand the biophysics of target searching and develop rational approaches for mitigating off-target activity while accounting for the influence of torsional strain in the genome. Here we investigate the dynamics of Cas9 R-loop formation and collapse using rotor bead tracking (RBT), a single-molecule technique that can simultaneously monitor DNA unwinding with base-pair resolution and binding of fluorescently labeled macromolecules in real time. By measuring changes in torque upon unwinding of the double helix, we find that R-loop formation and collapse proceed via a transient discrete intermediate, consistent with DNA:RNA hybridization within an initial seed region. Using systematic measurements of target and off-target sequences under controlled mechanical perturbations, we characterize position-dependent effects of sequence mismatches and show how DNA supercoiling modulates the energy landscape of R-loop formation and dictates access to states competent for stable binding and cleavage. Consistent with this energy landscape model, in bulk experiments we observe promiscuous cleavage under physiological negative supercoiling. The detailed description of DNA interrogation presented here suggests strategies for improving the specificity and kinetics of Cas9 as a genome engineering tool and may inspire expanded applications that exploit sensitivity to DNA supercoiling

Human THO–Sin3A interaction reveals new mechanisms to prevent R-loops that cause genome instability

R-loops, formed by co-transcriptional DNA–RNA hybrids and a displaced DNA single strand (ssDNA), fulfill certain positive regulatory roles but are also a source of genomic instability. One key cellular mechanism to prevent R-loop accumulation centers on the conserved THO/TREX complex, an RNA-binding factor involved in transcription elongation and RNA export that contributes to messenger ribonucleoprotein (mRNP) assembly, but whose precise function is still unclear. To understand how THO restrains harmful R-loops, we searched for new THO-interacting factors. We found that human THO interacts with the Sin3A histone deacetylase complex to suppress co-transcriptional R-loops, DNA damage, and replication impairment. Functional analyses show that histone hypo-acetylation prevents accumulation of harmful R-loops and RNA-mediated genomic instability. Diminished histone deacetylase activity in THO- and Sin3A-depleted cell lines correlates with increased R-loop formation, genomic instability, and replication fork stalling. Our study thus uncovers physical and functional crosstalk between RNA-binding factors and chromatin modifiers with a major role in preventing R-loop formation and RNA-mediated genome instability.European Research Council ERC2014 AdG669898 TARLOOPMinisterio de Economía y Competitividad BFU2013-42918-P, BFU2016-75058-PJunta de Andalucía BIO123

RNase H enables efficient repair of R-loop induced DNA damage.

R-loops, three-stranded structures that form when transcripts hybridize to chromosomal DNA, are potent agents of genome instability. This instability has been explained by the ability of R-loops to induce DNA damage. Here, we show that persistent R-loops also compromise DNA repair. Depleting endogenous RNase H activity impairs R-loop removal in Saccharomyces cerevisiae, causing DNA damage that occurs preferentially in the repetitive ribosomal DNA locus (rDNA). We analyzed the repair kinetics of this damage and identified mutants that modulate repair. We present a model that the persistence of R-loops at sites of DNA damage induces repair by break-induced replication (BIR). This R-loop induced BIR is particularly susceptible to the formation of lethal repair intermediates at the rDNA because of a barrier imposed by RNA polymerase I

The Fanconi Anemia Pathway Protects Genome Integrity from R-loops

Co-transcriptional RNA-DNA hybrids (R loops) cause genome instability. To prevent harmful R loop accumulation, cells have evolved specific eukaryotic factors, one being the BRCA2 double-strand break repair protein. As BRCA2 also protects stalled replication forks and is the FANCD1 member of the Fanconi Anemia (FA) pathway, we investigated the FA role in R loop-dependent genome instability. Using human and murine cells defective in FANCD2 or FANCA and primary bone marrow cells from FANCD2 deficient mice, we show that the FA pathway removes R loops, and that many DNA breaks accumulated in FA cells are R loop-dependent. Importantly, FANCD2 foci in untreated and MMC-treated cells are largely R loop dependent, suggesting that the FA functions at R loop-containing sites. We conclude that co-transcriptional R loops and R loop-mediated DNA damage greatly contribute to genome instability and that one major function of the FA pathway is to protect cells from R loops

Attenuation of RNA polymerase II pausing mitigates BRCA1-associated R-loop accumulation and tumorigenesis.

Most BRCA1-associated breast tumours are basal-like yet originate from luminal progenitors. BRCA1 is best known for its functions in double-strand break repair and resolution of DNA replication stress. However, it is unclear whether loss of these ubiquitously important functions fully explains the cell lineage-specific tumorigenesis. In vitro studies implicate BRCA1 in elimination of R-loops, DNA-RNA hybrid structures involved in transcription and genetic instability. Here we show that R-loops accumulate preferentially in breast luminal epithelial cells, not in basal epithelial or stromal cells, of BRCA1 mutation carriers. Furthermore, R-loops are enriched at the 5' end of those genes with promoter-proximal RNA polymerase II (Pol II) pausing. Genetic ablation of Cobra1, which encodes a Pol II-pausing and BRCA1-binding protein, ameliorates R-loop accumulation and reduces tumorigenesis in Brca1-knockout mouse mammary epithelium. Our studies show that Pol II pausing is an important contributor to BRCA1-associated R-loop accumulation and breast cancer development

Interplay between DNA sequence and negative superhelicity drives R-loop structures.

R-loops are abundant three-stranded nucleic-acid structures that form in cis during transcription. Experimental evidence suggests that R-loop formation is affected by DNA sequence and topology. However, the exact manner by which these factors interact to determine R-loop susceptibility is unclear. To investigate this, we developed a statistical mechanical equilibrium model of R-loop formation in superhelical DNA. In this model, the energy involved in forming an R-loop includes four terms-junctional and base-pairing energies and energies associated with superhelicity and with the torsional winding of the displaced DNA single strand around the RNA:DNA hybrid. This model shows that the significant energy barrier imposed by the formation of junctions can be overcome in two ways. First, base-pairing energy can favor RNA:DNA over DNA:DNA duplexes in favorable sequences. Second, R-loops, by absorbing negative superhelicity, partially or fully relax the rest of the DNA domain, thereby returning it to a lower energy state. In vitro transcription assays confirmed that R-loops cause plasmid relaxation and that negative superhelicity is required for R-loops to form, even in a favorable region. Single-molecule R-loop footprinting following in vitro transcription showed a strong agreement between theoretical predictions and experimental mapping of stable R-loop positions and further revealed the impact of DNA topology on the R-loop distribution landscape. Our results clarify the interplay between base sequence and DNA superhelicity in controlling R-loop stability. They also reveal R-loops as powerful and reversible topology sinks that cells may use to nonenzymatically relieve superhelical stress during transcription

Histone Mutants Separate R Loop Formation from Genome Instability Induction

R loops have positive physiological roles, but they can also be deleterious by causing genome instability, and the mechanisms for this are unknown. Here we identified yeast histone H3 and H4 mutations that facilitate R loops but do not cause instability. R loops containing single-stranded DNA (ssDNA), versus RNA-DNA hybrids alone, were demonstrated using ssDNA-specific human AID and bisulfite. Notably, they are similar size regardless of whether or not they induce genome instability. Contrary to mutants causing R loop-mediated instability, these histone mutants do not accumulate H3 serine-10 phosphate (H3S10-P). We propose a two-step mechanism in which, first, an altered chromatin facilitates R loops, and second, chromatin is modified, including H3S10-P, as a requisite for compromising genome integrity. Consistently, these histone mutations suppress the high H3S10 phosphorylation and genomic instability of hpr1 and sen1 mutants. Therefore, contrary to what was previously believed, R loops do not cause genome instability by themselves.European Research Council ERC2014 AdG669898Ministerio de Economía y Competitividad BFU2013-42918-P, BFU2016-75058-

Single-Molecule Insight Into Target Recognition by CRISPR–Cas Complexes

Ribonucleoprotein (RNP) complexes from CRISPR–Cas systems have attracted enormous interest since they can be easily and flexibly reprogrammed to target any desired locus for genome engineering and gene regulation applications. Basis for the programmability is a short RNA (crRNA) inside these complexes that recognizes the target nucleic acid by base pairing. For CRISPR–Cas systems that target double-stranded DNA this results in local DNA unwinding and formation of a so-called R-loop structure. Here we provide an overview how this target recognition mechanism can be dissected in great detail at the level of a single molecule. Specifically, we demonstrate how magnetic tweezers are applied to measure the local DNA unwinding at the target in real time. To this end we introduce the technique and the measurement principle. By studying modifications of the consensus target sequence, we show how different sequence elements contribute to the target recognition mechanism. From these data, a unified target recognition mechanism can be concluded for the RNPs Cascade and Cas9 from types I and II CRISPR–Cas systems. R-loop formation is hereby initiated on the target at an upstream element, called protospacer adjacent motif (PAM), from which the R-loop structure zips directionally toward the PAM-distal end of the target. At mismatch positions, the R-loop propagation stalls and further propagation competes with collapse of the structure. Upon full R-loop zipping conformational changes within the RNPs trigger degradation of the DNA target. This represents a shared labor mechanism in which zipping between nucleic acid strands is the actual target recognition mechanism while sensing of the R-loop arrival at the PAM-distal end just verifies the success of the full zipping