TULIPs decorate the three-dimensional genome of PFA ependymoma.

Posterior fossa group A (PFA) ependymoma is a lethal brain cancer diagnosed in infants and young children. The lack of driver events in the PFA linear genome led us to search its 3D genome for characteristic features. Here, we reconstructed 3D genomes from diverse childhood tumor types and uncovered a global topology in PFA that is highly reminiscent of stem and progenitor cells in a variety of human tissues. A remarkable feature exclusively present in PFA are type B ultra long-range interactions in PFAs (TULIPs), regions separated by great distances along the linear genome that interact with each other in the 3D nuclear space with surprising strength. TULIPs occur in all PFA samples and recur at predictable genomic coordinates, and their formation is induced by expression of EZHIP. The universality of TULIPs across PFA samples suggests a conservation of molecular principles that could be exploited therapeutically.

CRISPR-based genome editing through the lens of DNA repair.

Genome editing technologies operate by inducing site-specific DNA perturbations that are resolved by cellular DNA repair pathways. Products of genome editors include DNA breaks generated by CRISPR-associated nucleases, base modifications induced by base editors, DNA flaps created by prime editors, and integration intermediates formed by site-specific recombinases and transposases associated with CRISPR systems. Here, we discuss the cellular processes that repair CRISPR-generated DNA lesions and describe strategies to obtain desirable genomic changes through modulation of DNA repair pathways. Advances in our understanding of the DNA repair circuitry, in conjunction with the rapid development of innovative genome editing technologies, promise to greatly enhance our ability to improve food production, combat environmental pollution, develop cell-based therapies, and cure genetic and infectious diseases.

CRISPR-Mediated Base Editing Enables Efficient Disruption of Eukaryotic Genes through Induction of STOP Codons.

Standard CRISPR-mediated gene disruption strategies rely on Cas9-induced DNA double-strand breaks (DSBs). Here, we show that CRISPR-dependent base editing efficiently inactivates genes by precisely converting four codons (CAA, CAG, CGA, and TGG) into STOP codons without DSB formation. To facilitate gene inactivation by induction of STOP codons (iSTOP), we provide access to a database of over 3.4 million single guide RNAs (sgRNAs) for iSTOP (sgSTOPs) targeting 97%-99% of genes in eight eukaryotic species, and we describe a restriction fragment length polymorphism (RFLP) assay that allows the rapid detection of iSTOP-mediated editing in cell populations and clones. To simplify the selection of sgSTOPs, our resource includes annotations for off-target propensity, percentage of isoforms targeted, prediction of nonsense-mediated decay, and restriction enzymes for RFLP analysis. Additionally, our database includes sgSTOPs that could be employed to precisely model over 32,000 cancer-associated nonsense mutations. Altogether, this work provides a comprehensive resource for DSB-free gene disruption by iSTOP.

Acetylation of PCNA Sliding Surface by Eco1 Promotes Genome Stability through Homologous Recombination.

During DNA replication, proliferating cell nuclear antigen (PCNA) adopts a ring-shaped structure to promote processive DNA synthesis, acting as a sliding clamp for polymerases. Known posttranslational modifications function at the outer surface of the PCNA ring to favor DNA damage bypass. Here, we demonstrate that acetylation of lysine residues at the inner surface of PCNA is induced by DNA lesions. We show that cohesin acetyltransferase Eco1 targets lysine 20 at the sliding surface of the PCNA ring in vitro and in vivo in response to DNA damage. Mimicking constitutive acetylation stimulates homologous recombination and robustly suppresses the DNA damage sensitivity of mutations in damage tolerance pathways. In comparison to the unmodified trimer, structural differences are observed at the interface between protomers in the crystal structure of the PCNA-K20ac ring. Thus, acetylation regulates PCNA sliding on DNA in the presence of DNA damage, favoring homologous recombination linked to sister-chromatid cohesion.

Phospho-dependent recruitment of the yeast NuA4 acetyltransferase complex by MRX at DNA breaks regulates RPA dynamics during resection.

The KAT5 (Tip60/Esa1) histone acetyltransferase is part of NuA4, a large multifunctional complex highly conserved from yeast to mammals that targets lysines on H4 and H2A (X/Z) tails for acetylation. It is essential for cell viability, being a key regulator of gene expression, cell proliferation, and stem cell renewal and an important factor for genome stability. The NuA4 complex is directly recruited near DNA double-strand breaks (DSBs) to facilitate repair, in part through local chromatin modification and interplay with 53BP1 during the DNA damage response. While NuA4 is detected early after appearance of the lesion, its precise mechanism of recruitment remains to be defined. Here, we report a stepwise recruitment of yeast NuA4 to DSBs first by a DNA damage-induced phosphorylation-dependent interaction with the Xrs2 subunit of the Mre11-Rad50-Xrs2 (MRX) complex bound to DNA ends. This is followed by a DNA resection-dependent spreading of NuA4 on each side of the break along with the ssDNA-binding replication protein A (RPA). Finally, we show that NuA4 can acetylate RPA and regulate the dynamics of its binding to DNA, hence targeting locally both histone and nonhistone proteins for lysine acetylation to coordinate repair.

Precise deposition of histone H2A.Z in chromatin for genome expression and maintenance.

Histone variant H2A.Z is essential in higher eukaryotes and has different functions in the cell. Several studies indicate that H2A.Z is found at specific loci in the genome such as regulatory-gene regions, where it poises genes for transcription. Itsdeposition creates chromatin regions with particular structural characteristics which could favor rapid transcription activation. This review focuses on the highly regulated mechanism of H2A.Z deposition in chromatin which is essential for genome integrity. Chaperones escort H2A.Z to large ATP-dependent chromatin remodeling enzymes which are responsible for its deposition/eviction. Over the last ten years, biochemical, genetic and genomic studies helped us understand the precise role of these complexes in this process. It hasbeen suggested that a cooperation occurs between histone acetyltransferase and chromatin remodeling activities to incorporate H2A.Z in chromatin. Its regulated deposition near centromeres and telomeres also shows its implication in chromosomal structure integrity and parallels a role in DNA damage response. Thedynamics of H2A.Z deposition/eviction at specific loci was shown to be critical for genome expression andmaintenance, thus cell fate. Altogether, recent findings reassert the importance of the regulated depositionof this histone variant. This article is part of a Special Issue entitled: Histone chaperones and Chromatin assembly.

One-pot DTECT enables rapid and efficient capture of genetic signatures for precision genome editing and clinical diagnostics.

The detection of genomic sequences and their alterations is crucial for basic research and clinical diagnostics. However, current methodologies are costly and time-consuming and require outsourcing sample preparation, processing, and analysis to genomic companies. Here, we establish One-pot DTECT, a platform that expedites the detection of genetic signatures, only requiring a short incubation of a PCR product in an optimized one-pot mixture. One-pot DTECT enables qualitative, quantitative, and visual detection of biologically relevant variants, such as cancer mutations, and nucleotide changes introduced by prime editing and base editing into cancer cells and human primary T cells. Notably, One-pot DTECT achieves quantification accuracy for targeted genetic signatures comparable with Sanger and next-generation sequencing. Furthermore, its effectiveness as a diagnostic platform is demonstrated by successfully detecting sickle cell variants in blood and saliva samples. Altogether, One-pot DTECT offers an efficient, versatile, adaptable, and cost-effective alternative to traditional methods for detecting genomic signatures.

A detection method for the capture of genomic signatures: From disease diagnosis to genome editing.

Variations in the genetic information originate from errors during DNA replication, error-prone repair of DNA damages, or genome editing. The most common approach to detect changes in DNA sequences employs sequencing technologies. However, they remain expensive and time-consuming, limiting their utility for routine laboratory experiments. We recently developed DinucleoTidE Signature CapTure (DTECT). DTECT is a marker-free and versatile detection method that captures targeted dinucleotide signatures resulting from the digestion of genomic amplicons by the type IIS restriction enzyme AcuI. Here, we describe the DTECT protocol to identify mutations introduced by CRISPR-based precision genome editing technologies or resulting from genetic variation. DTECT enables accurate detection of mutations using basic laboratory equipment and off-the-shelf reagents with qualitative or quantitative capture of signatures.

Detection of Marker-Free Precision Genome Editing and Genetic Variation through the Capture of Genomic Signatures.

Genome editing technologies have transformed our ability to engineer desired genomic changes within living systems. However, detecting precise genomic modifications often requires sophisticated, expensive, and time-consuming experimental approaches. Here, we describe DTECT (Dinucleotide signaTurE CapTure), a rapid and versatile detection method that relies on the capture of targeted dinucleotide signatures resulting from the digestion of genomic DNA amplicons by the type IIS restriction enzyme AcuI. DTECT enables the accurate quantification of marker-free precision genome editing events introduced by CRISPR-dependent homology-directed repair, base editing, or prime editing in various biological systems, such as mammalian cell lines, organoids, and tissues. Furthermore, DTECT allows the identification of oncogenic mutations in cancer mouse models, patient-derived xenografts, and human cancer patient samples. The ease, speed, and cost efficiency by which DTECT identifies genomic signatures should facilitate the generation of marker-free cellular and animal models of human disease and expedite the detection of human pathogenic variants.

Stimulation of CRISPR-mediated homology-directed repair by an engineered RAD18 variant.

Precise editing of genomic DNA can be achieved upon repair of CRISPR-induced DNA double-stranded breaks (DSBs) by homology-directed repair (HDR). However, the efficiency of this process is limited by DSB repair pathways competing with HDR, such as non-homologous end joining (NHEJ). Here we individually express in human cells 204 open reading frames involved in the DNA damage response (DDR) and determine their impact on CRISPR-mediated HDR. From these studies, we identify RAD18 as a stimulator of CRISPR-mediated HDR. By defining the RAD18 domains required to promote HDR, we derive an enhanced RAD18 variant (e18) that stimulates CRISPR-mediated HDR in multiple human cell types, including embryonic stem cells. Mechanistically, e18 induces HDR by suppressing the localization of the NHEJ-promoting factor 53BP1 to DSBs. Altogether, this study identifies e18 as an enhancer of CRISPR-mediated HDR and highlights the promise of engineering DDR factors to augment the efficiency of precision genome editing.

Novel mechanism of PCNA control through acetylation of its sliding surface.

Recent findings revealed a new unexpected regulatory mechanism that controls the proliferating cell nuclear antigen (PCNA). Multiple positively-charged lysine residues located on the ring inner surface are neutralized by acetylation and required for cellular resistance to Desoxyribonucleic acid (DNA) damage. Here, we summarize the key observations, discuss implications, and perspectives linked to cancer, as well as challenges for future work.