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PubMed2025

SelectRepair Knockout: Efficient PTC-Free Gene Knockout Through Selectable Homology-Directed DNA Repair.

Generating nonessential gene knockouts using CRISPR/Cas9 technology is becoming increasingly common in biological research. In a typical workflow, the Cas9 endonuclease is used to induce a DNA double-strand break that relies on nonhomologous end-joining (NHEJ) to introduce a premature termination codon (PTC) in the target gene. The goal is to isolate clones in which the gene produces PTC-containing mRNA transcripts that are degraded via nonsense-mediated mRNA decay (NMD) to cause loss of gene function. Unfortunately, this approach is laborious, and not all PTCs trigger NMD. More importantly, mounting evidence suggest that PTC mutations can also result in a transcriptional adaptation response that can mask the effects of a PTC-mediated gene knockout. In this chapter, we present a PTC-free gene knockout strategy that implements homology-directed DNA repair (HDR) with selectable markers to substantially reduce the complexity of the screening and validation of genome edits in cells containing more than one gene copy as in the case of the commonly used hypotriploid HEK293 cell line. We describe how to obtain a complete knockout of the Ligase IV protein (LIG4) and provide considerations for the application of this SelectRepair Knockout method to other genes.

Humans↗

PubMed2026

BCDX2-CX3 and DX2-CX3 complexes assemble and stabilize RAD51 filaments.

The repair of DNA double-strand breaks by homologous recombination is essential for genomic integrity, and its dysregulation is a hallmark of cancer1. Central to homologous recombination is the RAD51 recombinase, whose assembly into a nucleoprotein filament is governed by five RAD51 paralogues (RAD51B, RAD51C, RAD51D, XRCC2 and XRCC3)2. Mutations in any of these proteins predispose individuals to multiple cancers or genetic disorders3-6. These paralogues are thought to form two functionally separate complexes RAD51B-RAD51C-RAD51D-XRCC2 (BCDX2) and RAD51C-XRCC3 (CX3), that act independently at different stages of homologous recombination7-11. Here we demonstrate that all five paralogues can assemble into a single, ATP-dependent BCDX2-CX3-RAD51 supercomplex. The architecture of this assembly bound to single-stranded DNA reveals a contiguous filament where the CX3 module stacks atop BCDX2, creating a protofilament template for RAD51 filament formation. We further identify a novel, RAD51B-independent DX2-CX3 complex (RAD51D-XRCC2-RAD51C-XRCC3) functioning as a stable RAD51 anchor on single-stranded DNA, and we capture it in multiple states, including capping RAD51 filament segment. These distinct assemblies are differentially regulated by ATPase activity, defining a dynamic BCDX2-CX3 'loader' and a stable DX2-CX3 'anchor' that provide functional modularity to the homologous recombination machinery. This work provides a unifying mechanism for human RAD51 paralogue function and delivers an atomic blueprint for interpreting disease-causing mutations.

Rad51 Recombinase↗

PubMed2025

Does chromoanagenesis play a role in the origin of B chromosomes?

B chromosomes (Bs) exist in addition to the standard (A) chromosomes in a wide range of species. The process underlying their origin is still unclear. We propose pathways of intra- and interspecific origin of B chromosomes based on known mechanisms of chromosome evolution and available knowledge of their sequence composition in different species. We speculate that a mitotic or meiotic segregation error of one or more A chromosomes initiates, via chromoanagenesis, the formation of a proto-B chromosome. In the second step, proto-B chromosomes accumulate A chromosome- and organelle-derived sequences over time, most likely via DNA double-strand break (DSB) mis-repair. Consequently, the original structure of the early stage proto-B chromosomes becomes masked by continuous sequence incorporation. The similarity between A chromosome sequences integrated into B chromosomes and the original sequences on the donor chromosomes decreases over time if there is no selection pressure on these sequences on B chromosomes. However, besides chromoanagenesis, also other mechanisms leading to the formation of B chromosomes might exist.

Evolution, Molecular↗

PubMed2026

CROP: a feature-independent context-aware method for CRISPR-Cas9 frameshift prediction.

MOTIVATION: The CRISPR-Cas9 complex has revolutionized genome-editing technologies. By designing a 20 nt-long guide RNA, a Cas9 nuclease can be guided to cleave almost any genomic target site (followed by NGG). The cleavage induces double-stranded DNA breaks, which are then repaired by cellular pathways. Accurate CRISPR-Cas9 repair-outcome prediction is essential for designing guide RNAs with desired genomic effects, such as gene knockout. A central challenge is quantifying the rate of frameshifts, i.e. repair-outcomes that lead to a change in the local length that is not a multiple of three. Previous methods for frameshift-rate prediction were trained on only a few experimental or cellular contexts, mostly relied on manually defined microhomology features, and were limited by sparse features and class labels. RESULTS: We developed CROP, a feature-independent context-aware repair-outcome prediction method. By aggregating specific repair outcomes as Δlength classes, CROP overcomes class sparsity. We designed CROP to work with variable input sequence lengths and output classes to utilize multiple datasets simultaneously. We benchmarked CROP against state-of-the-art repair-outcome prediction methods over 18 datasets, which we curated and standardized from various studies. Across all datasets, CROP outperformed all competing methods in frameshift-rate prediction. We performed cross-experiment and cross-cellular frameshift-rate predictions to investigate the generalizability of repair mechanisms. Finally, we show that CROP learned microhomology principles from raw sequences without explicit feature engineering, establishing an end-to-end architecture for CRISPR-Cas9 repair-outcome prediction that learns from multiple datasets. AVAILABILITY AND IMPLEMENTATION: CROP is available at https://github.com/OrensteinLab/CROP.

CRISPR-Cas Systems↗

PubMed2026

Mechanisms of Hexavalent Chromium-Induced Reproductive Toxicity: A Focus on the Ovary and Placenta.

Hexavalent Chromium (Cr(VI)) is a Group A carcinogen, mutagen, and teratogen. Cr(VI) has been used by more than 50 industries, and its contamination of drinking water is widespread across the United States (U.S.). Epidemiological data of women who lived in Willits, California, U.S., indicate that environmental exposure to Cr(VI) adversely affects pregnancy outcomes and the health of their immediate offspring, resulting in a low birth rate, pregnancy loss, and spontaneous abortion, and their children (F1 offspring) experienced birth defects. However, the molecular mechanisms behind Cr(VI)-induced reproductive and developmental toxicity are poorly understood. Cr(VI) enters cells through anion transporters and is rapidly reduced to Cr(III) by endogenous antioxidants within the cell. Cr(III) forms adducts with DNA, which can block DNA replication and transcription; abnormal repair can lead to DNA double-strand breaks, mutations, micronucleus formation, chromosomal abnormalities, and increased genomic instability. Cr(VI) induces oxidative stress via the Fenton reaction, generating free radicals, and depleting antioxidants, thereby promoting apoptosis via p53-dependent and independent pathways, resulting in follicular atresia and accelerated reproductive aging. Antioxidant supplementation with resveratrol, vitamin C, and edaravone mitigates Cr(VI) toxicity in the ovary. Cr(VI) disrupts meiosis in metaphase II oocytes by causing DNA strand breaks, altering F-actin dynamics, disturbing microtubules, and leading to chromosome missegregation. Gestational exposure to Cr(VI) also disrupts placental function through multiple mechanisms by targeting trophoblast lineages. The current review focuses on genotoxicity, oxidative stress, and other mechanisms by which Cr(VI) disrupts the female reproductive and endocrine systems, with particular emphasis on the ovary and placenta.

Hexavalent chromium↗

PubMed2026

Prime assembly with linear DNA donors enables large genomic insertions.

Targeted insertion of large DNA fragments has promising applications for genome engineering and gene therapy1,2. Twin prime-editing guide RNAs have enabled relatively large insertions, but the efficiency remains low for insertions greater than 400 base pairs3-6. Here we describe a prime assembly (PA) approach for the insertion of large DNA donor fragments, of which the ends are designed to overlap with the flaps generated by twin prime editing (twinPE). We used PA to insert one or multiple overlapping DNA fragments, with total insertion sizes ranging from 0.1 kb to 11 kb. An inhibitor of non-homologous end joining enhanced both the efficiency and precision of insertions. PA relies on DNA templates that are easily produced, does not require co-delivery of exogenous DNA-dependent DNA polymerases and proceeds in non-cycling cells, suggesting independence from canonical homology-directed repair pathways. Our study demonstrates that PA can initiate Gibson-like assembly in cells to generate gene insertions without double-stranded DNA breaks, recombinases or homology-directed repair.

Animals↗

PubMed2025

Distinct roles of the two BRCA2 DNA-binding domains in DNA damage repair and replication fork preservation.

Homologous recombination (HR) removes DNA double-strand breaks (DSBs) and preserves stressed DNA replication forks. Successful HR execution requires the tumor suppressor BRCA2, which harbors distinct DNA-binding domains (DBDs): one that possesses three oligonucleotide/oligosaccharide-binding (OB) folds (OB-DBD) and another residing in the C-terminal recombinase binding domain (CTRB-DBD). Here, we employ multi-faceted approaches to delineate the contributions of these domains toward HR and replication fork maintenance. We show that OB-DBD and CTRB-DBD confer single-strand DNA (ssDNA)- and dsDNA-binding capabilities, respectively, and that BRCA2 variants mutated in either domain are impaired in their ability to load the recombinase RAD51 onto ssDNA pre-occupied by RPA. While the CTRB-DBD mutant is modestly affected by DNA break repair, it exhibits a strong defect in the protection of stressed replication forks. In contrast, the OB-DBD is indispensable for both BRCA2 functions. Our study thus defines the unique contributions of the two BRCA2 DBDs in genome maintenance.

BRCA2 Protein↗

PubMed2026

An Update on Inborn Errors of V(D)J Recombination.

V(D)J recombination is the fundamental process by which developing T and B lymphocytes generate diverse antigen receptors, enabling adaptive immunity. This tightly regulated program operates exclusively in lymphoid precursors during G1 phase and depends on the lymphocyte-specific RAG1-RAG2 recombinase to introduce programmed DNA double-strand breaks at recombination signal sequences, followed by repair through the classical nonhomologous end joining (c-NHEJ) pathway. Disruption of any step in this molecular choreography compromises antigen receptor diversity and underlies a spectrum of inborn errors of immunity (IEIs), ranging from severe combined immunodeficiency (SCID) to immune dysregulation with autoimmunity and granulomatous disease. In this review, we place disorders of V(D)J recombination within the broader framework of T-cell development, detailing the temporal waves of recombinase activity, chromatin accessibility, and DNA damage responses that guide thymocyte differentiation. We discuss pathogenic variants affecting the cleavage phase [RAG1, RAG2, and the recently identified RAG cochaperone NudC domain-containing 3 (NUDCD3)], end processing (ARTEMIS), ligation and repair (LIG4, XLF, XRCC4, PRKDC), and genome surveillance pathways (ATM, MRN complex, RNF168), highlighting genotype-phenotype correlations and mechanisms driving immune deficiency and dysregulation. We briefly review recent diagnostic advances, including newborn screening using T-cell receptor excision circles, repertoire sequencing, and functional assays, alongside current therapeutic strategies. Finally, we outline key unanswered questions and argue that continued integration of clinical observation with molecular discovery is essential to improve outcomes and deepen understanding of adaptive immune development.

Humans↗

PubMed2026

Advances in CRISPR Base Editing: From Molecular Evolution to Therapeutic Applications in Genomic Medicine.

CRISPR-Cas9 systems revolutionized gene editing, but inherent drawbacks, namely DNA double-strand breaks (DSBs) and the difficulty of achieving precise repairs (due to low HDR efficiency), led researchers to invent new, more accurate gene editing tools. Base editing represents a significant leap forward, enabling targeted single-nucleotide conversions directly on the DNA without DSBs or donor templates. The core technology involves fusing catalytically dead or nickase Cas proteins to DNA deaminase enzymes. Cytosine base editors (CBEs) convert C•G to T•A pairs, while adenine base editors (ABEs) change A•T to G•C. These editors exploit the deaminase function within the R-loop structure formed by Cas binding and co-opt endogenous DNA repair mechanisms for precision. While offering improved efficiency and editing precision, base editing faces persistent challenges, such as off-target effects, bystander edits, delivery and ethical concerns. Continuous engineering efforts have refined these tools, enhancing accuracy, expanding targetability and reducing unwanted edits. The base editing arsenal has also broadened to include C-to-G base editors (CGBEs), dual A&C editors and versions targeting organelles. Successful preclinical studies demonstrating the correction of mutations responsible for the disease have paved the way for clinical trials, which are now testing therapies for conditions like sickle cell disease, β-thalassaemia and hypercholesterolemia using various delivery systems. This review explores CRISPR base editing's origins, mechanisms of action, potential therapies and current restrictions, pointing to its broadening impact on medical genetics.

Humans↗

PubMed2026

RAD54L coordinates the nucleolar DNA damage response to maintain rDNA stability.

The nucleolus is organized around actively transcribed ribosomal RNA genes (rDNA), where high RNA polymerase I (Pol I) activity creates intrinsic susceptibility to replication stress and DNA damage. Here, we identify the DNA translocase RAD54L as a critical regulator of the nucleolar DNA damage response (nDDR) to rDNA double-strand breaks (DSBs) and replication stress. We show that RAD54L localizes to the nucleolus under basal conditions and is recruited to nucleolar caps following CRISPR-Cas9-induced rDNA-DSBs to promote repair. RAD54L loss results in persistent RAD51 foci, increased nucleolar γH2AX, and micronuclei formation, indicating defective resolution of rDNA lesions and genome instability. Under baseline conditions and replication stress induced by the Pol I transcription inhibitor CX-5461, RAD54L limits the accumulation of ssDNA and coordinates nDDR signaling. We further show that rDNA-DSBs induce RNA polymerase II-dependent RNA-DNA hybrids (R-loops) at intergenic rDNA regions, which facilitate nucleolar reorganization and cap formation and repair factor recruitment. Together, these findings establish RAD54L as a key regulator that coordinates replication stress response and rDNA repair, maintaining rDNA stability and genome integrity.

DNA, Ribosomal↗

PubMed2026

SET domain bifurcated histone lysine methyltransferase 1 regulates histone modification and DNA damage response during zygotic genome activation in pigs.

SET domain bifurcated histone lysine methyltransferase 1 (SETDB1) is a key epigenetic regulator that catalyzes histone H3 lysine 9 trimethylation (H3K9me3), a mark essential for transcriptional repression and heterochromatin formation. Here, we investigated the role of SETDB1 during zygotic genome activation (ZGA) in porcine embryos. SETDB1 knockdown (KD) was induced by microinjecting double-stranded RNA (dsRNA), and its impact on early embryonic development was evaluated. SETDB1 KD decreased H3K9me3 levels, markedly increased H3K9ac, and downregulated ZGA-associated genes. These epigenetic alterations were accompanied by impaired cleavage, reduced blastocyst formation, and a lower total cell number. Upon etoposide-induced DNA double-strand breaks, SETDB1 KD embryos showed reduced expression of key DNA repair proteins, failed to efficiently restore DNA integrity, and exhibited increased apoptosis, indicating a compromised DNA damage response and repair process. SETDB1 KD also reduced HDAC3 expression, suggesting that SETDB1 may regulate HDAC3 to maintain histone acetylation balance. Consistently, HDAC3 inhibition increased H3K9ac, decreased H3K9me3, and reduced SETDB1 protein levels, supporting a reciprocal regulatory relationship. Together, these findings indicate that SETDB1 is important for porcine embryonic development by coordinating histone modifications and safeguarding genomic integrity during ZGA, and they suggest that the interplay between SETDB1 and HDAC3 constitutes a potentially important epigenetic axis for proper histone modification dynamics and developmental competence.

Animals↗

PubMed2025

Rapid Generation of Recombinant Poxviruses Using CRISPR/Cas9 Gene Editing.

The low-frequency natural recombination that is detected in poxvirus-infected cells has long been used to genetically modify poxviruses. Such recombinant poxviruses have found many applications as vaccines for preventing infectious diseases and as experimental cancer therapeutics. Unfortunately, these methods are time consuming, can leave behind "scars" or selectable markers, and many months of work may be required to generate plaque-purified recombinants bearing multiple virus gene substitutions, deletions, and/or inserted transgenes. Over the last decade, several reports have described how CRISPR/Cas9 technologies can be used to better facilitate genetic manipulation of vaccinia virus (VACV). These protocols use Cas9/gRNA complexes to introduce double-stranded breaks into specific sites in virus genomic DNA either in vivo or in vitro. Recombination-repair reactions are then employed to repair the breaks using transfected DNAs encoding the required homologies and desired mutation(s). Here we describe a method where we combine CRISPR/Cas9 genome editing in vitro, followed by Leporipoxvirus-catalyzed repair and reactivation of the cut VACV DNA using repair fragments provided in trans. This method optimizes several steps in the preparation of the CRISPR/Cas9-cut VACV DNA and can be used to introduce mutations at multiple sites without requiring selectable markers. It also provides some guidance regarding how the position of the CRISPR/Cas9-cuts can affect co-conversion of flanking markers embedded in the repair fragment. The method allows researchers to quickly generate recombinant VACV bearing multiple genetic alterations and using only a single round of reactivation and plating.

CRISPR-Cas Systems↗

PubMed2026

MRE11 suppresses germline mutagenesis at meiotic double-strand breaks in mice.

SPO11 forms hundreds of double-strand breaks (DSBs) to initiate meiotic recombination that is normally error-free. However, SPO11 activity can be mutagenic when one chromatid incurs closely spaced DSBs (double cuts), especially when DSBs are dysregulated by loss of the ATM kinase. De novo indels and structural variants can arise via end joining at double cuts within a single hotspot (microdeletions) or at adjacent hotspots separated by at least 30 kb, as we now show, sometimes accompanied by ectopic insertions of double-cut fragments. Here, we investigate how meiotic DSB end processing influences end joining. In MRE11-deficient mouse spermatocytes, which do not resect their DSBs, deletions at double cuts occur readily, with end-joining breakpoint profiles closely matching SPO11 DSB profiles. Microdeletions suggest that two DSBs can be as close as ∼21 bp. The tyrosyl-DNA phosphodiesterase TDP2 contributes to both deletion formation and ectopic insertion of double-cut fragments, presumably by removing SPO11 from DNA ends prior to joining. Finally, observations suggest a cooperative role for MRE11 and ATM in locally regulating DSB distributions. Our findings provide insight into the mechanism of de novo mutation origin, emphasizing the role of meiotic DSBs in shaping genome evolution.

Animals↗

PubMed2026

Maternal DNA repair safeguards genome stability during the oocyte-to-embryo transition.

De novo mutations are a major source of genetic variation and disease risk, yet the developmental timing and mechanisms underlying their origin require further investigation. While germ cells have traditionally been considered the primary source of these mutations, increasing evidence suggests that a substantial fraction arise after fertilization. Here, we investigated the role of maternal DNA repair in shaping mutagenesis during this critical window by using a mouse model with oocyte-specific disruption of the homologous recombination factor RAD51 and a combination of cellular and molecular analyses. Loss of maternal RAD51 led to the accumulation of DNA double-strand breaks in oocytes without impairing their growth, meiotic maturation, or fertilization competence. In contrast, embryos derived from RAD51-deficient oocytes exhibited increased DNA damage and developmental delay during early cleavage stages. Whole-genome sequencing revealed a significant increase in de novo variants in offspring, the majority displaying intermediate allele frequencies consistent with post-zygotic mosaic mutations. These findings confirm that maternal DNA repair safeguards genome stability across the oocyte-to-embryo transition and identify early embryogenesis as a major source of de novo mutations, with implications for reproductive biology and the origins of genetic diseases.

DNA↗

PubMed2026

A dual-dimensional CRISPR toolkit enables one-step high-efficiency multiplex genome editing in Komagataella phaffii.

Against the backdrop of green biomanufacturing, engineering methanol-utilizing Komagataella phaffii (K. phaffii) represents an effective strategy to expand the one carbon (C1) product profile and speed up the industrialization of C1-based bioeconomy. To address the technical challenges of low efficiency and cumbersome experimental procedures for multiplex gene editing and precise large-fragment integration during the reconstruction of complex metabolic pathways in K. phaffii, this study established a CRISPR toolkit - Efficient Multi-Gene Editing System 3.0 (EMGES 3.0) - which enabled one-step large-fragment integration coupled with multiplex gene knockout. EMGES 3.0 was constructed through the synergistic optimization of a repair-engineered chassis and an episomal CRISPR vector. For chassis engineering, five DNA repair modules: Δlig4 (DNA Ligase IV, non-homologous end joining end ligation), ppMRE11(The endogenous MRE11 gene from Pichia pastoris) overexpression (The Meiotic Recombination 11, DNA double-strand break end resection), Δrad9 (Radiation-Sensitive 9, DNA damage checkpoint regulation), Δmph1 (Mutator Phenotype Helicase 1, improvement of homologous recombinant strand extension), and PapRecT-PaSSB co-expression (stabilization of recombination intermediates) were integrated to generate the highly recombinogenic strain Y09. For vector engineering, cenARS was replaced by panARS and the endogenous promoter PGAP was employed to drive the double hammerhead ribozyme-single guide RNA-hepatitis delta virus ribozyme (double HH-sgRNA-HDV: dHgH)-mediated sgRNA expression, yielding the optimized vector Nov_pGAP_panARS_pLAT1_Cas9. These two features on K. phaffii together enhanced the EMGES 3.0 to a higher standard of transformation rate and editing efficiency. According to our results, EMGES 3.0 achieved dual-functional gene knockout efficiencies between 76.6% and 100%. For insertion of medium-long fragments (>4.5 kb), the efficiency achieved 93.3%. In addition, the one-step integration of ultra-long fragments (>16 kb) achieved 14.8%, which was reported for the first time. Furthermore, the efficiency of simultaneous long-fragment integration at three neutral loci reached 38.4% (>15 kb). We applied the system for one-step production of free fatty acids (FFAs, yield: 5.82 ∼ 7.30 mg/L/OD600) and resveratrol (yield: 1.14 ∼ 1.28 mg/L) using methanol as the sole carbon source. EMGES 3.0 provides a robust technical foundation for complex compounds biosynthesis and high-yield industrial strains, while also advancing K. phaffii as an industrial synthetic biology chassis for efficient C1 utilization.

CRISPR-Cas Systems↗

PubMed2026

Dynamic chromatin tethering of MDC1 regulates genome stability.

DNA double-strand breaks (DSBs) are highly cytotoxic DNA lesions because they disrupt the connectivity of the DNA strand. Homologous recombination (HR) is a high-fidelity DSB repair pathway that copies the sequence spanning the break from a homologous template, but how DNA ends are held together during HR remains unclear. Here we demonstrate that the proline-serine-threonine (PST) repeat region of Mediator of DNA Damage Checkpoint 1 (MDC1) is a multivalent nucleosome-binding domain, sufficient to tether chromatin in multiple contexts. In interphase, the constitutive chromatin association of MDC1 is critical for RAD51 loading and efficient HR. In mitosis, PST-mediated chromatin binding is attenuated by phosphorylation, preventing aberrant chromosomal interactions while preserving DNA tethering by the MDC1-TOPBP1-CIP2A complex. In total, this work demonstrates that the PST repeat region of MDC1 is a multivalent nucleosome-binding domain with tunable affinity that supports DSB repair by HR and maintains genome stability during mitosis.

Genomic Instability↗

PubMed2026

Linker Histone H1 Phosphorylation Promotes DNA Damage Repair during Replication Stress.

DNA replication fidelity depends on the integrity of the replication fork to prevent DNA damage and preserve genome stability. Disruptions to this process can trigger replication stress, leading to the accumulation of single-strand DNA (ssDNA) and double-strand breaks (DSBs), which drive mutagenesis and ultimately contribute to disease. While the roles of core histones and their post-translational modifications in this context have been more well-studied, far less is known about how linker histones regulate the replication stress response. Here, we demonstrate that the S-phase-phosphorylated form of the linker histone H1 (pH1) plays a key role in DNA damage repair at collapsed replication forks, both in vitro and in cells. Using phosphomimic and phosphonull H1 mutants, we show that phosphorylation enhances H1 assembly with ssDNA. Utilizing intein chemistry for the site-specific incorporation of a photocrosslinker to the C-terminus of H1, we map the direct interactors of H1. We identify phosphorylated H1 at replication forks, where it engages replication machinery and DNA damage response factors, including Histone PARylation Factor 1 (HPF1). We further demonstrate that ssDNA induces pH1-HPF1 interactions that promote liquid-like assemblies, correlating with reduced DNA damage and histone PARylation. Consistent with this role, reduction of total H1 increases cellular sensitivity to DNA damage, a phenotype that is partially rescued by reintroduction of H1.4. Together, these findings establish pH1 as a new regulator of DNA damage repair at collapsed replication forks through the controlled sequestration of repair factors.

Journal Article↗

PubMed2026

Isolation of region-specific factors driving antibody class-switch recombination from the immunoglobulin heavy chain locus.

Activation-Induced Cytidine Deaminase (AID) induces DNA double-strand breaks (DSBs) at the switch (S) regions of the Immunoglobulin heavy chain (IgH) locus, which are essential for class switch recombination (CSR) and somatic hypermutation (SHM), key processes for effective antibody production. While AID activity is critical, its off-target effects, such as DSBs at the Myc locus, can cause chromosomal translocations like IgH-Myc fusions, contributing to B-cell lymphomas. The factors assembled on the IgH locus that help restrict AID-induced DSBs and subsequently CSR, remain unknown. To address this, we developed a method to isolate CSR-specific factors by inserting a 5×-GAL4-UAS sequence at the switch-mu (Sμ) region in CH12 cells. This engineered site enables recruitment of a 3-FLAG-GAL4 DNA-binding protein (3F-GAL4-DBD), allowing specific pulldown of proteins enriched at the Sμ region. Successful recovery of the known CSR regulator BRD2 from the Sμ region, along with enrichment of the DNA repair factors 53BP1 and gH2AX, validated this approach. Identification and characterization of IgH-enriched factors establish a validated methodological framework to facilitate future proteomic discovery of CSR regulators and highlight mechanisms that balance antibody diversification with genomic integrity in B cells.

Immunoglobulin Class Switching↗