Chromatin-centric insights into DNA replication.

DNA replication ensures the precise transmission of genetic information from parent to daughter cells. In eukaryotes, this process involves the replication of every base pair within a highly complex chromatin environment, encompassing multiple levels of chromatin structure and various chromatin metabolic processes. Recent evidence has demonstrated that DNA replication is strictly regulated in both temporal and spatial dimensions by factors such as 3D genome structure and transcription, which is crucial for maintaining genomic stability in each cell cycle.

Transfer of mitochondrial DNA into the nuclear genome during induced DNA breaks.

Mitochondria serve as the cellular powerhouse, and their distinct DNA makes them a prospective target for gene editing to treat genetic disorders. However, the impact of genome editing on mitochondrial DNA (mtDNA) stability remains a mystery. Our study reveals previously unknown risks of genome editing that both nuclear and mitochondrial editing cause discernible transfer of mitochondrial DNA segments into the nuclear genome in various cell types including human cell lines, primary T cells, and mouse embryos.

DNA polymerase δ subunit Pol32 binds histone H3-H4 and couples nucleosome assembly with Okazaki fragment processing.

During lagging strand chromatin replication, multiple Okazaki fragments (OFs) require processing and nucleosome assembly, but the mechanisms linking these processes remain unclear. Here, using transmission electron microscopy and rapid degradation of DNA ligase Cdc9, we observed flap structures accumulated on lagging strands, controlled by both Pol δ's strand displacement activity and Fen1's nuclease digestion. The distance between neighboring flap structures exhibits a regular pattern, indicative of matured OF length.

Fork coupling directs DNA replication elongation and termination.

DNA replication is initiated at multiple loci to ensure timely duplication of eukaryotic genomes. Sister replication forks progress bidirectionally, and replication terminates when two convergent forks encounter one another. To investigate the coordination of replication forks, we developed a replication-associated in situ HiC method to capture chromatin interactions involving nascent DNA.

Cohesin maintains replication timing to suppress DNA damage on cancer genes.

Cohesin loss-of-function mutations are frequently observed in tumors, but the mechanism underlying its role in tumorigenesis is unclear. Here, we found that depletion of RAD21, a core subunit of cohesin, leads to massive genome-wide DNA breaks and 147 translocation hotspot genes, co-mutated with cohesin in multiple cancers. Increased DNA damages are independent of RAD21-loss-induced transcription alteration and loop anchor disruption.

Human 8-cell embryos enable efficient induction of disease-preventive mutations without off-target effect by cytosine base editor.

Approximately 140 million people worldwide are homozygous carriers of APOE4 (ε4), a strong genetic risk factor for late onset familial and sporadic Alzheimer's disease (AD), 91% of whom will develop AD at earlier age than heterozygous carriers and noncarriers. Susceptibility to AD could be reduced by targeted editing of APOE4, but a technical basis for controlling the off-target effects of base editors is necessary to develop low-risk personalized gene therapies. Here, we first screened eight cytosine base editor variants at four injection stages (from 1- to 8-cell stage), and found that FNLS-YE1 variant in 8-cell embryos achieved the comparable base conversion rate (up to 100%) with the lowest bystander effects.

Safeguarding genome integrity during gene-editing therapy in a mouse model of age-related macular degeneration.

Ensuring genome safety during gene editing is crucial for clinical translation of the high-efficient CRISPR-Cas9 toolbox. Therefore, the undesired events including chromosomal translocations, vector integrations, and large deletions arising during therapeutic gene editing remain to be adequately addressed or tackled in vivo. Here, we apply CRISPR-Cas9TX in comparison to CRISPR-Cas9 to target Vegfa for the treatment of age-related macular degeneration (AMD) disease in a mouse model.

Engineering of efficiency-enhanced Cas9 and base editors with improved gene therapy efficacies.

Editing efficiency is pivotal for the efficacies of CRISPR-based gene therapies. We found that fusing an HMG-D domain to the N terminus of SpCas9 (named efficiency-enhanced Cas9 [eeCas9]) significantly increased editing efficiency by 1.4-fold on average.

CRISPR/Cas9-induced structural variations expand in T lymphocytes in vivo.

CRISPR/Cas9 has been adapted to disrupt endogenous genes in adoptive T-lymphocyte therapy to prevent graft-versus-host disease. However, genome editing also generates prevalent deleterious structural variations (SVs), including chromosomal translocations and large deletions, raising safety concerns about reinfused T cells. Here, we dynamically monitored the progression of SVs in a mouse model of T-cell receptor (TCR)-transgenic T-cell adoptive transfer, mimicking TCR T therapeutics.

Comprehensive assessment of miniature CRISPR-Cas12f nucleases for gene disruption.

Because of their small size, the recently developed CRISPR-Cas12f nucleases can be effectively packaged into adeno-associated viruses for gene therapy. However, a systematic evaluation of the editing outcomes of CRISPR-Cas12f is lacking. In this study, we apply a high-throughput sequencing method to comprehensively assess the editing efficiency, specificity, and safety of four Cas12f proteins in parallel with that of Cas9 and two Cas12a proteins at multiple genomic sites.

CTCF DNA-binding domain undergoes dynamic and selective protein-protein interactions.

CTCF is a predominant insulator protein required for three-dimensional chromatin organization. However, the roles of its insulation of enhancers in a 3D nuclear organization have not been fully explained. Here, we found that the CTCF DNA-binding domain (DBD) forms dynamic self-interacting clusters.

Long-term correction of hemophilia B through CRISPR/Cas9 induced homology-independent targeted integration.

CRISPR/Cas9-mediated site-specific insertion of exogenous genes holds potential for clinical applications. However, it is still infeasible because homologous recombination (HR) is inefficient, especially for non-dividing cells. To overcome the challenge, we report that a homology-independent targeted integration (HITI) strategy is used for permanent integration of high-specificity-activity Factor IX variant (F9 Padua, R338L) at the albumin (Alb) locus in a novel hemophilia B (HB) rat model.

The origin of unwanted editing byproducts in gene editing.

The rapid development of CRISPR-Cas genome editing tools has greatly changed the way to conduct research and holds tremendous promise for clinical applications. During genome editing, CRISPR-Cas enzymes induce DNA breaks at the target sites and subsequently the DNA repair pathways are recruited to generate diverse editing outcomes. Besides off-target cleavage, unwanted editing outcomes including chromosomal structural variations and exogenous DNA integrations have recently raised concerns for clinical safety.

C-terminal deletion-induced condensation sequesters AID from IgH targets in immunodeficiency.

In activated B cells, activation-induced cytidine deaminase (AID) generates programmed DNA lesions required for antibody class switch recombination (CSR), which may also threaten genome integrity. AID dynamically shuttles between cytoplasm and nucleus, and the majority stays in the cytoplasm due to active nuclear export mediated by its C-terminal peptide. In immunodeficient-patient cells expressing mutant AID lacking its C-terminus, a catalytically active AID-delC protein accumulates in the nucleus but nevertheless fails to support CSR.

PEM-seq comprehensively quantifies DNA repair outcomes during gene-editing and DSB repair.

The repair products of double-stranded DNA breaks (DSBs) are crucial for investigating the mechanism underlying DNA damage repair as well as evaluating the safety and efficiency of gene-editing; however, a comprehensively quantitative assay remains to be established. Here, we describe the step-by-step instructions of the primer extension-mediated sequencing (PEM-seq), followed by the framework of data processing and statistical analysis. PEM-seq presents a full spectrum of repair outcomes for both genome-editing-induced and endogenous DSBs in mouse and human cells.

Cas9 exo-endonuclease eliminates chromosomal translocations during genome editing.

The mechanism underlying unwanted structural variations induced by CRISPR-Cas9 remains poorly understood, and no effective strategy is available to inhibit the generation of these byproducts. Here we find that the generation of a high level of translocations is dependent on repeated cleavage at the Cas9-targeting sites. Therefore, we employ a strategy in which Cas9 is fused with optimized TREX2 to generate Cas9TX, a Cas9 exo-endonuclease, which prevents perfect DNA repair and thereby avoids repeated cleavage.

Improving the efficiency of CRISPR-Cas12a-based genome editing with site-specific covalent Cas12a-crRNA conjugates.

The CRISPR-Cas12a system shows unique features compared with widely used Cas9, making it an attractive and potentially more precise alternative. However, the adoption of this system has been hindered by its relatively low editing efficiency. Guided by physical chemical principles, we covalently conjugated 5' terminal modified CRISPR RNA (crRNA) to a site-specifically modified Cas12a through biorthogonal chemical reaction.

RAG2 abolishes RAG1 aggregation to facilitate V(D)J recombination.

RAG1 and RAG2 form a tetramer nuclease to initiate V(D)J recombination in developing T and B lymphocytes. The RAG1 protein evolves from a transposon ancestor and possesses nuclease activity that requires interaction with RAG2. Here, we show that the human RAG1 aggregates in the nucleus in the absence of RAG2, exhibiting an extremely low V(D)J recombination activity.

Global detection of DNA repair outcomes induced by CRISPR-Cas9.

CRISPR-Cas9 generates double-stranded DNA breaks (DSBs) to activate cellular DNA repair pathways for genome editing. The repair of DSBs leads to small insertions or deletions (indels) and other complex byproducts, including large deletions and chromosomal translocations. Indels are well understood to disrupt target genes, while the other deleterious byproducts remain elusive.

In-depth assessment of the PAM compatibility and editing activities of Cas9 variants.

A series of Cas9 variants have been developed to improve the editing fidelity or targeting range of CRISPR-Cas9. Here, we employ a high-throughput sequencing approach primer-extension-mediated sequencing to analyze the editing efficiency, specificity and protospacer adjacent motif (PAM) compatibility of a dozen of SpCas9 variants at multiple target sites in depth, and our findings validate the high fidelity or broad editing range of these SpCas9 variants. With regard to the PAM-flexible SpCas9 variants, we detect significantly increased levels of off-target activity and propose a trade-off between targeting range and editing specificity for them, especially for the near-PAM-less SpRY.

Transcription shapes DNA replication initiation to preserve genome integrity.

Background: Early DNA replication occurs within actively transcribed chromatin compartments in mammalian cells, raising the immediate question of how early DNA replication coordinates with transcription to avoid collisions and DNA damage.

Results: We develop a high-throughput nucleoside analog incorporation sequencing assay and identify thousands of early replication initiation zones in both mouse and human cells. The identified early replication initiation zones fall in open chromatin compartments and are mutually exclusive with transcription elongation.

FACT interacts with Set3 HDAC and fine-tunes GAL1 transcription in response to environmental stimulation.

The histone chaperone facilitates chromatin transactions (FACT) functions in various DNA transactions. How FACT performs these multiple functions remains largely unknown. Here, we found, for the first time, that the N-terminal domain of its Spt16 subunit interacts with the Set3 histone deacetylase complex (Set3C) and that FACT and Set3C function in the same pathway to regulate gene expression in some settings.

Structural insight into the assembly and conformational activation of human origin recognition complex.

The function of the origin recognition complex (ORC) in DNA replication is highly conserved in recognizing and marking the initiation sites. The detailed molecular mechanisms by which human ORC is reconfigured into a state competent for origin association remain largely unknown. Here, we present structural characterizations of human ORC1-5 and ORC2-5 assemblies.