Conformational control of Cas9 by CRISPR hybrid RNA-DNA guides mitigates off-target activity in T cells.

The off-target activity of the CRISPR-associated nuclease Cas9 is a potential concern for therapeutic genome editing applications. Although high-fidelity Cas9 variants have been engineered, they exhibit varying efficiencies and have residual off-target effects, limiting their applicability. Here, we show that CRISPR hybrid RNA-DNA (chRDNA) guides provide an effective approach to increase Cas9 specificity while preserving on-target editing activity. Across multiple genomic targets in primary human T cells, we show that 2'-deoxynucleotide (dnt) positioning affects guide activity and specificity in a target-dependent manner and that this can be used to engineer chRDNA guides with substantially reduced off-target effects. Crystal structures of DNA-bound Cas9-chRDNA complexes reveal distorted guide-target duplex geometry and allosteric modulation of Cas9 conformation. These structural effects increase specificity by perturbing DNA hybridization and modulating Cas9 activation kinetics to disfavor binding and cleavage of off-target substrates. Overall, these results pave the way for utilizing customized chRDNAs in clinical applications.

Multilayered mechanisms ensure that short chromosomes recombine in meiosis.

In most species, homologous chromosomes must recombine in order to segregate accurately during meiosis1. Because small chromosomes would be at risk of missegregation if recombination were randomly distributed, the double-strand breaks (DSBs) that initiate recombination are not located arbitrarily2. How the nonrandomness of DSB distributions is controlled is not understood, although several pathways are known to regulate the timing, location and number of DSBs. Meiotic DSBs are generated by Spo11 and accessory DSB proteins, including Rec114 and Mer2, which assemble on chromosomes3-7 and are nearly universal in eukaryotes8-11. Here we demonstrate how Saccharomyces cerevisiae integrates multiple temporally distinct pathways to regulate the binding of Rec114 and Mer2 to chromosomes, thereby controlling the duration of a DSB-competent state. The engagement of homologous chromosomes with each other regulates the dissociation of Rec114 and Mer2 later in prophase I, whereas the timing of replication and the proximity to centromeres or telomeres influence the accumulation of Rec114 and Mer2 early in prophase I. Another early mechanism enhances the binding of Rec114 and Mer2 specifically on the shortest chromosomes, and is subject to selection pressure to maintain the hyperrecombinogenic properties of these chromosomes. Thus, the karyotype of an organism and its risk of meiotic missegregation influence the shape and evolution of its recombination landscape. Our results provide a cohesive view of a multifaceted and evolutionarily constrained system that allocates DSBs to all pairs of homologous chromosomes.

Ensuring meiotic DNA break formation in the mouse pseudoautosomal region.

Sex chromosomes in males of most eutherian mammals share only a small homologous segment, the pseudoautosomal region (PAR), in which the formation of double-strand breaks (DSBs), pairing and crossing over must occur for correct meiotic segregation1,2. How cells ensure that recombination occurs in the PAR is unknown. Here we present a dynamic ultrastructure of the PAR and identify controlling cis- and trans-acting factors that make the PAR the hottest segment for DSB formation in the male mouse genome. Before break formation, multiple DSB-promoting factors hyperaccumulate in the PAR, its chromosome axes elongate and the sister chromatids separate. These processes are linked to heterochromatic mo-2 minisatellite arrays, and require MEI4 and ANKRD31 proteins but not the axis components REC8 or HORMAD1. We propose that the repetitive DNA sequence of the PAR confers unique chromatin and higher-order structures that are crucial for recombination. Chromosome synapsis triggers collapse of the elongated PAR structure and, notably, oocytes can be reprogrammed to exhibit spermatocyte-like levels of DSBs in the PAR simply by delaying or preventing synapsis. Thus, the sexually dimorphic behaviour of the PAR is in part a result of kinetic differences between the sexes in a race between the maturation of the PAR structure, formation of DSBs and completion of pairing and synapsis. Our findings establish a mechanistic paradigm for the recombination of sex chromosomes during meiosis.

DNA Repair Profiling Reveals Nonrandom Outcomes at Cas9-Mediated Breaks.

The repair outcomes at site-specific DNA double-strand breaks (DSBs) generated by the RNA-guided DNA endonuclease Cas9 determine how gene function is altered. Despite the widespread adoption of CRISPR-Cas9 technology to induce DSBs for genome engineering, the resulting repair products have not been examined in depth. Here, the DNA repair profiles of 223 sites in the human genome demonstrate that the pattern of DNA repair following Cas9 cutting at each site is nonrandom and consistent across experimental replicates, cell lines, and reagent delivery methods. Furthermore, the repair outcomes are determined by the protospacer sequence rather than genomic context, indicating that DNA repair profiling in cell lines can be used to anticipate repair outcomes in primary cells. Chemical inhibition of DNA-PK enabled dissection of the DNA repair profiles into contributions from c-NHEJ and MMEJ. Finally, this work elucidates a strategy for using "error-prone" DNA-repair machinery to generate precise edits.

Allogeneic chimeric antigen receptor-T cells with CRISPR-disrupted programmed death-1 checkpoint exhibit enhanced functional fitness.

BACKGROUND AIMS: Therapeutic disruption of immune checkpoints has significantly advanced the armamentarium of approaches for treating cancer. The prominent role of the programmed death-1 (PD-1)/programmed death ligand-1 axis for downregulating T cell function offers a tractable strategy for enhancing the disease-modifying impact of CAR-T cell therapy. METHODS: To address checkpoint interference, primary human T cells were genome edited with a next-generation CRISPR-based platform (Cas9 chRDNA) by knockout of the PDCD1 gene encoding the PD-1 receptor. Site-specific insertion of a chimeric antigen receptor specific for CD19 into the T cell receptor alpha constant locus was implemented to drive cytotoxic activity. RESULTS: These allogeneic CAR-T cells (CB-010) promoted longer survival of mice in a well-established orthotopic tumor xenograft model of a B cell malignancy compared with identically engineered CAR-T cells without a PDCD1 knockout. The persistence kinetics of CB-010 cells in hematologic tissues versus CAR-T cells without PDCD1 disruption were similar, suggesting the robust initial debulking of established tumor xenografts was due to enhanced functional fitness. By single-cell RNA-Seq analyses, CB-010 cells, when compared with identically engineered CAR-T cells without a PDCD1 knockout, exhibited fewer Treg cells, lower exhaustion phenotypes and reduced dysfunction signatures and had higher activation, glycolytic and oxidative phosphorylation signatures. Further, an enhancement of mitochondrial metabolic fitness was observed, including increased respiratory capacity, a hallmark of less differentiated T cells. CONCLUSIONS: Genomic PD-1 checkpoint disruption in the context of allogeneic CAR-T cell therapy may provide a compelling option for treating B lymphoid malignancies.

Apollo contributes to G overhang maintenance and protects leading-end telomeres.

Mammalian telomeres contain a single-stranded 3' overhang that is thought to mediate telomere protection. Here we identify the TRF2-interacting factor Apollo as a nuclease that contributes to the generation/maintenance of this overhang. The function of mouse Apollo was determined using Cre-mediated gene deletion, complementation with Apollo mutants, and the TRF2-F120A mutant that cannot bind Apollo. Cells lacking Apollo activated the ATM kinase at their telomeres in S phase and showed leading-end telomere fusions. These telomere dysfunction phenotypes were accompanied by a reduction in the telomeric overhang signal. The telomeric functions of Apollo required its TRF2-interaction and nuclease motifs. Thus, TRF2 recruits the Apollo nuclease to process telomere ends synthesized by leading-strand DNA synthesis, thereby creating a terminal structure that avoids ATM activation and resists end-joining. These data establish that the telomeric overhang is required for the protection of telomeres from the DNA damage response.

Meiotic recombination initiation in and around retrotransposable elements in Saccharomyces cerevisiae.

Meiotic recombination is initiated by large numbers of developmentally programmed DNA double-strand breaks (DSBs), ranging from dozens to hundreds per cell depending on the organism. DSBs formed in single-copy sequences provoke recombination between allelic positions on homologous chromosomes, but DSBs can also form in and near repetitive elements such as retrotransposons. When they do, they create a risk for deleterious genome rearrangements in the germ line via recombination between non-allelic repeats. A prior study in budding yeast demonstrated that insertion of a Ty retrotransposon into a DSB hotspot can suppress meiotic break formation, but properties of Ty elements in their most common physiological contexts have not been addressed. Here we compile a comprehensive, high resolution map of all Ty elements in the rapidly and efficiently sporulating S. cerevisiae strain SK1 and examine DSB formation in and near these endogenous retrotransposable elements. SK1 has 30 Tys, all but one distinct from the 50 Tys in S288C, the source strain for the yeast reference genome. From whole-genome DSB maps and direct molecular assays, we find that DSB levels and chromatin structure within and near Tys vary widely between different elements and that local DSB suppression is not a universal feature of Ty presence. Surprisingly, deletion of two Ty elements weakened adjacent DSB hotspots, revealing that at least some Ty insertions promote rather than suppress nearby DSB formation. Given high strain-to-strain variability in Ty location and the high aggregate burden of Ty-proximal DSBs, we propose that meiotic recombination is an important component of host-Ty interactions and that Tys play critical roles in genome instability and evolution in both inbred and outcrossed sexual cycles.

Apollo, an Artemis-related nuclease, interacts with TRF2 and protects human telomeres in S phase.

Human chromosome ends are protected by shelterin, an abundant six-subunit protein complex that binds specifically to the telomeric-repeat sequences, regulates telomere length, and ensures that chromosome ends do not elicit a DNA-damage response (reviewed in). Using mass spectrometry of proteins associated with the shelterin component Rap1, we identified an SMN1/PSO2 nuclease family member that is closely related to Artemis. We refer to this protein as Apollo and report that Apollo has the ability to localize to telomeres through an interaction with the shelterin component TRF2. Although its low abundance at telomeres indicates that Apollo is not a core component of shelterin, Apollo knockdown with RNAi resulted in senescence and the activation of a DNA-damage signal at telomeres as evidenced by telomere-dysfunction-induced foci (TIFs). The TIFs occurred primarily in S phase, suggesting that Apollo contributes to a processing step associated with the replication of chromosome ends. Furthermore, some of the metaphase chromosomes showed two telomeric signals at single-chromatid ends, suggesting an aberrant telomere structure. We propose that the Artemis-like nuclease Apollo is a shelterin accessory factor required for the protection of telomeres during or after their replication.

The entire Nup107-160 complex, including three new members, is targeted as one entity to kinetochores in mitosis.

In eukaryotes, bidirectional transport of macromolecules between the cytoplasm and the nucleus occurs through elaborate supramolecular structures embedded in the nuclear envelope, the nuclear pore complexes (NPCs). NPCs are composed of multiple copies of approximately 30 different proteins termed nucleoporins, of which several can be biochemically isolated as subcomplexes. One such building block of the NPC, termed the Nup107-160 complex in vertebrates, was so far demonstrated to be composed of six different nucleoporins. Here, we identify three WD (Trp-Asp)-repeat nucleoporins as new members of this complex, two of which, Nup37 and Nup43, are specific to higher eukaryotes. The third new member Seh1 is more loosely associated with the Nup107-160 complex biochemically, but its depletion by RNA interference leads to phenotypes similar to knock down of other constituents of this complex. By combining green fluorescent protein-tagged nucleoporins and specific antibodies, we show that all the constituents of this complex, including Nup37, Nup43, Seh1, and Sec13, are targeted to kinetochores from prophase to anaphase of mitosis. Together, our results indicate that the entire Nup107-160 complex, which comprises nearly one-third of the so-far identified nucleoporins, specifically localizes to kinetochores in mitosis.

Loss of Rap1 induces telomere recombination in the absence of NHEJ or a DNA damage signal.

Shelterin is an essential telomeric protein complex that prevents DNA damage signaling and DNA repair at mammalian chromosome ends. Here we report on the role of the TRF2-interacting factor Rap1, a conserved shelterin subunit of unknown function. We removed Rap1 from mouse telomeres either through gene deletion or by replacing TRF2 with a mutant that does not bind Rap1. Rap1 was dispensable for the essential functions of TRF2--repression of ATM kinase signaling and nonhomologous end joining (NHEJ)--and mice lacking telomeric Rap1 were viable and fertile. However, Rap1 was critical for the repression of homology-directed repair (HDR), which can alter telomere length. The data reveal that HDR at telomeres can take place in the absence of DNA damage foci and underscore the functional compartmentalization within shelterin.

A shared docking motif in TRF1 and TRF2 used for differential recruitment of telomeric proteins.

Mammalian telomeres are protected by a six-protein complex: shelterin. Shelterin contains two closely related proteins (TRF1 and TRF2), which recruit various proteins to telomeres. We dissect the interactions of TRF1 and TRF2 with their shared binding partner (TIN2) and other shelterin accessory factors. TRF1 recognizes TIN2 using a conserved molecular surface in its TRF homology (TRFH) domain. However, this same surface does not act as a TIN2 binding site in TRF2, and TIN2 binding to TRF2 is mediated by a region outside the TRFH domain. Instead, the TRFH docking site of TRF2 binds a shelterin accessory factor (Apollo), which does not interact with the TRFH domain of TRF1. Conversely, the TRFH domain of TRF1, but not of TRF2, interacts with another shelterin-associated factor: PinX1.

TIN2 binds TRF1 and TRF2 simultaneously and stabilizes the TRF2 complex on telomeres.

Human telomeres contain two related telomeric DNA-binding proteins, TRF1 and TRF2. The TRF1 complex contains the TRF1 interacting partner, TIN2, as well as PIP1 and POT1 and regulates telomere-length homeostasis. The TRF2 complex is primarily involved in telomere protection and contains the TRF2 interacting partner human (h)Rap1 as well as several factors involved in the DNA damage response. A prior report showed that conditional deletion of murine TRF1 reduced the presence of TRF2 on telomeres. Here we showed that TRF2 is also lost from human telomeres upon TRF1 depletion with small interfering RNA prompting a search for the connection between the TRF1 and TRF2 complexes. Using mass spectrometry and co-immunoprecipitation, we found that TRF1, TIN2, PIP1, and POT1 are associated with the TRF2-hRap1 complex. Gel filtration identified a TRF2 complex containing TIN2 and POT1 but not TRF1 indicating that TRF1 is not required for this interaction. Co-immunoprecipitation, Far-Western assays, and two-hybrid assays showed that TIN2, but not POT1 or PIP1, interacts directly with TRF2. Furthermore, TIN2 was found to bind TRF1 and TRF2 simultaneously, showing that TIN2 can link these telomeric proteins. This connection appeared to stabilize TRF2 on the telomeres as the treatment of cells with TIN2 small interfering RNA resulted in a decreased presence of TRF2 and hRap1 at chromosome ends. The TIN2-mediated cooperative binding of TRF1 and TRF2 to telomeres has important implications for the mechanism of telomere length regulation and protection.