Homology-directed Repair Research Articles

Human PC4 supports telomere stability and viability in cells utilizing the alternative lengthening of telomeres mechanism.

Cancer cells with an activated Alternative Lengthening of Telomeres (ALT) mechanism elongate telomeres via homology-directed repair. Sustained telomeric replication stress is an essential trigger of ALT activity; however, it can lead to cell death if not properly restricted. By analyzing publicly available data from genome-wide CRISPR KO screenings, we have identified the multifunctional protein PC4 as a novel factor essential for ALT cell viability. Depletion of PC4 results in rapid ALT cell death, while telomerase-positive cells show minimal effects. PC4 depletion induces replication stress and telomere fragility primarily in ALT cells, and increases ALT activity. PC4 binds to telomeric DNA in cells, and its binding can be enhanced by telomeric replication stress. Finally, a mutant PC4 with partly impaired single stranded DNA binding activity is capable to localize to telomeres and suppress ALT activity and telomeric replication stress. We propose that PC4 supports ALT cell viability, at least partly, by averting telomere dysfunction. Further studies of PC4 interactions at ALT telomeres may hold promise for innovative therapies to eradicate ALT cancers.

CRISPR/Cas system-mediated base editing in crops: recent developments and future prospects.

Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 9 (CRISPR/Cas9) genome-editing system has altered plant research by allowing for targeted genome alteration, and they are emerging as powerful tools for evaluating plant gene function and improving crop yield. Even though CRISPR/Cas9 cleavage and subsequent repair are effective ways to precisely replace genes and change base pairs in plants, the dominance of the non-homologous end-joining pathway (NHEJ) and homology-directed repair's (HDR) poor effectiveness in plant cells have restricted their use. Base editing is gaining popularity as a potential alternative to HDR or NHEJ-mediated replacement, allowing for precise changes in the plant genome via programmed conversion of a single base to another without the need for a donor repair template or double-stranded breaks. In this review, we primarily present the mechanisms of base-editing system, including their distinct types such as DNA base editors (cytidine base editor and adenine base editor) and RNA base editors discovered so far. Next, we outline the current potential applications of the base-editing system for crop improvements. Finally, we discuss the limitations and potential future directions of the base-editing system in terms of improving crop quality. We hope that this review will enable the researcher to gain knowledge about base-editing tools and their potential applications in crop improvement.

Regulation of genome edited organismsin Australia.

Whether organisms developed with the use of genome editing techniques, or food derived from such organisms, are, or should be, regulated as genetically modified organisms (GMOs) or genetically modified (GM) food, respectively, remains a subject of debate globally. Much of the discussion has been scientific and focussed on the similar genetic outcomes of some genome editing techniques and 'conventional' or natural mutagenesis. Many jurisdictions, including Australia, have considered, or are considering, how their regulatory frameworks will deal with such organisms and products. In Australia, organisms developed with site directed nuclease 1 (SDN-1, with no added template to guide homology-directed repair) are not regulated as GMOs, pursuant to exclusions in the Gene Technology Regulations 2001. The exclusion of SDN-1 organismsfrom regulation in Australia is sometimes misrepresented, including in scientific peer reviewed publications, as extending to all genome edited organisms. This highlights the importance for researchers, developers and other stakeholders to understand that whether genome edited organisms are, or are not, subject to regulation as GMOs in a particular jurisdiction may quintessentially be a legal question, not a scientific one.

Germline variants of homology-directed repair or mismatch repair genes in cervical cancer.

While cervical cancer is associated with a persistent human papillomavirus (HPV) infection, the progression to cancer is influenced by genomic risk factors that have remained largely obscure. Pathogenic variants in genes of the homology-directed repair (HDR) or mismatch repair (MMR) are known to predispose to diverse tumour entities including breast and ovarian cancer (HDR) or colon and endometrial cancer (MMR). We here investigate the spectrum of HDR and MMR germline variants in cervical cancer, with particular focus on the HPV status and histological subgroups. We performed targeted next-generation sequencing for 5 MMR genes and 12 HDR genes on 728 German patients with cervical dysplasia or invasive cancer. In total, 4% of our patients carried a pathogenic germline variant, based on ClinVar classifications and additional ESM1b and AlphaMissense predictions. These included 15 patients with truncating variants in HDR genes (BARD1, BRCA1, BRCA2, BRIP1, FANCM, RAD51D and SLX4). MMR-related gene variants were less prevalent and mainly of the missense type. While MMR-related gene variants tended to associate with adenocarcinomas, HDR gene variants were commonly observed in squamous cancers. While one patient with HPV-negative cancer carried a pathogenic MMR gene variant (in MSH6), the HDR germline variants were found in patients with HPV-positive cancers and tended to associate with HPV18. Taken together, our study supports a potentially risk-modifying role of MMR and HDR germline variants in cervical cancer but no association with HPV-negative status. These variants may be exploitable in future therapeutic managements.

Introduction of asingle-nucleotide variant, rs16851030, into the ADORA1 gene increased cellular susceptibility to hypoxia.

Aim: Rs16851030, a single-nucleotide variant located in the 3'-untranslated region of the ADORA1 gene, has been proposed as a potential marker of caffeine sensitivity in apnea of prematurity. Besides, it is associated with aspirin-induced asthma and the development of acute chest syndrome. However, its functional significance is still unconfirmed. This study aimed to elucidate the functional impact of rs16851030 by using CRISPR/Cas9 approach to induce the DNA variant and attendant physiological changes.Methods: Rs16851030 was introduced into HEK293 cells via homology-directed repair (HDR). Edited cells were fluorescence-enriched, sorted, isolated, and expanded into single-cell-derived clones. The edit was confirmed by Sanger sequencing. RNA sequencing was used to analyze affected pathways.Results: Rs16851030-mutant cells showed increased susceptibility to hypoxia, a condition related to apnea of prematurity. After 24h of hypoxia, the viability of mutant clones 1 and 2 was low compared with wild-type cells (75.45% and 74.47% vs. 96.34%). RNA sequencing revealed transcriptomic changes linked to this increased vulnerability.Conclusion: Rs16851030 impairs cellular resistance to hypoxia, suggesting its role in conditions like apnea of prematurity. Further research should investigate the molecular mechanisms and transcriptomic alterations caused by rs16851030 under hypoxic conditions.

Molecular mechanisms underlying glucose-dependent insulinotropic polypeptide secretion in human duodenal organoids.

Glucose-dependent insulinotropic polypeptide (GIP) is an incretin hormone secreted by enteroendocrine K cells in the proximal small intestine. This study aimed to explore the function of human K cells at the molecular and cellular levels. CRISPR-Cas9 homology-directed repair was used to insert transgenes encoding a yellow fluorescent protein (Venus) or an Epac-based cAMP sensor (Epac-S-H187) in the GIP locus in human duodenal-derived organoids. Fluorescently labelled K cells were purified by FACS for RNA-seq and peptidomic analysis. GIP reporter organoids were employed for GIP secretion assays, live-cell imaging of Ca2+ using Fura-2 and cAMP using Epac-S-H187, and basic electrophysiological characterisation. The G protein-coupled receptor genes GPR142 and CASR were knocked out to evaluate roles in amino acid sensing. RNA-seq of human duodenal K cells revealed enrichment of several G protein-coupled receptors involved in nutrient sensing, including FFAR1, GPBAR1, GPR119, CASR and GPR142. Glucose induced action potential firing and cytosolic Ca2+ elevation and caused a 1.8-fold increase in GIP secretion, which was inhibited by the sodium glucose co-transporter 1/2 (SGLT1/2) blocker sotagliflozin. Activation of the long-chain fatty acid receptor free fatty acid receptor 1 (FFAR1) induced a 2.7-fold increase in GIP secretion, while tryptophan and phenylalanine stimulated secretion by 2.8- and 2.1-fold, respectively. While CASR knockout blunted intracellular Ca2+ responses, a CASR/GPR142 double knockout was needed to reduce GIP secretory responses to aromatic amino acids. The newly generated human organoid K cell model enables transcriptomic and functional characterisation of nutrient-sensing pathways involved in human GIP secretion. Both calcium-sensing receptor (CASR) and G protein-coupled receptor 142 (GPR142) contribute to protein-stimulated GIP secretion. This model will be further used to identify potential targets for modulation of native GIP secretion in diabetes and obesity.

CRISPR-Mediated Gene Editing Tool Development for Patients with Usher Syndrome Type II

Usher syndrome, a rare genetic disease affecting hearing and vision, is an autosomal recessive condition affecting 4 to 17 per 100,000 people and accounting for about 50 percent of hereditary deaf-blindness cases. Usher syndrome type II is caused by mutations of the USH2A gene, a 15.7 kb gene encoding the protein usherin, which localizes to photoreceptor cilium and cochlear hair cells. This subtype of Usher syndrome includes hearing loss from birth and progressive loss of vision, prompting retinitis pigmentosa (RP). Prime editing is a viable option for addressing USH2A mutations and restoring usherin expression and function. To begin, a HEK293T cell line needs to be established for subsequent testing of prime editors. Since prime editing to make the cell line was unsuccessful, homology-directed repair was done and successfully created a cell population with the mutation of interest (single base-pair deletion). Now that this cell population has been created, prime editors will be tested and optimized to address the mutation in a HEK293T cell line and potentially patient-derived induced pluripotent stem cells that are differentiated into photoreceptor precursor cells.

Lung and liver editing by lipid nanoparticle delivery of a stable CRISPR-Cas9 ribonucleoprotein.

Lipid nanoparticle (LNP) delivery of clustered regularly interspaced short palindromic repeat (CRISPR) ribonucleoproteins (RNPs) could enable high-efficiency, low-toxicity and scalable in vivo genome editing if efficacious RNP-LNP complexes can be reliably produced. Here we engineer a thermostable Cas9 from Geobacillus stearothermophilus (GeoCas9) to generate iGeoCas9 variants capable of >100× more genome editing of cells and organs compared with the native GeoCas9 enzyme. Furthermore, iGeoCas9 RNP-LNP complexes edit a variety of cell types and induce homology-directed repair in cells receiving codelivered single-stranded DNA templates. Using tissue-selective LNP formulations, we observe genome-editing levels of 16‒37% in the liver and lungs of reporter mice that receive single intravenous injections of iGeoCas9 RNP-LNPs. In addition, iGeoCas9 RNPs complexed to biodegradable LNPs edit the disease-causing SFTPC gene in lung tissue with 19% average efficiency, representing a major improvement over genome-editing levels observed previously using viral or nonviral delivery strategies. These results show that thermostable Cas9 RNP-LNP complexes can expand the therapeutic potential of genome editing.

The epistatic relationship of Drosophila melanogaster CtIP and Rif1 in homology-directed repair of DNA double-strand breaks.

Double-strand breaks (DSBs) are genotoxic DNA lesions that pose significant threats to genomic stability, necessitating precise and efficient repair mechanisms to prevent cell death or mutations. DSBs are repaired through nonhomologous end-joining (NHEJ) or homology-directed repair (HDR), which includes homologous recombination (HR) and single-strand annealing (SSA). CtIP and Rif1 are conserved proteins implicated in DSB repair pathway choice, possibly through redundant roles in promoting DNA end-resection required for HDR. Although the roles of these proteins have been well-established in other organisms, the role of Rif1 and its potential redundancies with CtIP in Drosophila melanogaster remain elusive. To examine the roles of DmCtIP and DmRif1 in DSB repair, this study employed the direct repeat of white (DR-white) assay, tracking across indels by decomposition (TIDE) analysis, and P{wIw_2 kb 3'} assay to track repair outcomes in HR, NHEJ, and SSA, respectively. These experiments were performed in DmCtIPΔ/Δ single mutants, DmRif1Δ/Δ single mutants, and DmRif1Δ/Δ; DmCtIPΔ/Δ double mutants. This work demonstrates significant defects in both HR and SSA repair in DmCtIPΔ/Δ and DmRif1Δ/Δ single mutants. However, experiments in DmRif1Δ/Δ; DmCtIPΔ/Δ double mutants reveal that DmCtIP is epistatic to DmRif1 in promoting HDR. Overall, this study concludes that DmRif1 and DmCtIP do not perform their activities in a redundant pathway, but rather DmCtIP is the main driver in promoting HR and SSA, most likely through its role in end resection.

Comparative sequencing study of mismatch repair and homology-directed repair genes in endometrial cancer and breast cancer patients from Kazakhstan.

Endometrial cancer has been associated with pathogenic variants in mismatch repair (MMR) genes, especially in the context of the hereditary Lynch Syndrome. More recently, pathogenic variants in genes of homology-directed repair (HDR) have also been suggested to contribute to a subset of endometrial cancers. In the present hospital-based study, we investigated the relative distribution of pathogenic MMR or HDR gene variants in a series of 342 endometrial cancer patients from the Oncology Clinic in Almaty, Kazakhstan. In comparison, we also sequenced 178 breast cancer patients from the same population with the same gene panel. Identified variants were classified according to ClinVar, ESM1b, and AlphaMissense prediction tools. We found 10 endometrial cancer patients (2.9%) carrying pathogenic or likely pathogenic variants in MMR genes (7 MSH6, 1 MSH2, 2 MUTYH), while 14 endometrial cancer patients (4.1%) carried pathogenic variants in HDR genes (4 BRCA2, 3 BRCA1, 3 FANCM, 2 SLX4, 1 BARD1, 1 BRIP1). In the breast cancer series, we found 8 carriers (4.5%) of pathogenic or likely pathogenic variants in MMR genes (2 MSH2, 2 MSH6, 4 MUTYH) while 12 patients (6.7%) harbored pathogenic or likely pathogenic HDR gene variants (5 BRCA1, 3 BRCA2, 1 BRIP1, 1 ERRC4, 1 FANCM, 1 SLX4). One patient who developed breast cancer first and endometrial cancer later carried a novel frameshift variant in MSH6. Our results indicate that MMR and HDR gene variants with predicted pathogenicity occur at substantial frequencies in both breast and endometrial cancer patients from the Kazakh population.

Functional Analysis of BARD1 Missense Variants on Homology-Directed Repair in Ovarian and Breast Cancers.

Women with germline BRCA1 mutations face an increased risk of developing breast and ovarian cancers. BARD1 (BRCA1 associated RING domain 1) is an essential heterodimeric partner of BRCA1, and mutations in BARD1 are also associated with these cancers. While BARD1 mutations are recognized for their cancer susceptibility, the exact roles of numerous BARD1 missense mutations remain unclear. In this study, we conducted functional assays to assess the homology-directed DNA repair (HDR) activity of all BARD1 missense substitutions identified in 55 breast and ovarian cancer samples, using the real-world data from the COSMIC and cBioPortal databases. Seven BARD1 variants (V85M, P187A, G491R, R565C, P669L, T719R, and Q730L) were confirmed to impair DNA damage repair. Furthermore, cells harboring these BARD1 variants exhibited increased sensitivity to the chemotherapeutic drugs, cisplatin, and olaparib, compared to cells expressing wild-type BARD1. These findings collectively suggest that these seven missense BARD1 variants are likely pathogenic and may respond well to cisplatin-olaparib combination therapy. This study not only enhances our understanding of BARD1's role in DNA damage repair but also offers valuable insights into predicting therapy responses in patients with specific BARD1 missense mutations.

Design and construction of a GAP-43 reporter system for potential identification of effective therapeutics for peripheral nerve regeneration

Background: Peripheral nerve injury (PNI) is a condition that can result in muscle paralysis and sensory disturbances. Electrical stimulation and/or the application of exogenous neurotrophic factors and cytokines are effective at enhancing nerve regeneration and is mediated via the expression of regeneration associated genes (RAGs) such as the growth associated protein GAP-43. Therapeutic upregulation of GAP-43 has potential use as a treatment for improving recovery from PNI. Few studies have investigated the potential of increasing GAP-43 for PNI therapeutic purposes and current methods for measuring GAP-43 expression are limited. Aims and Objectives: The broader aim of this work was to construct a motor neuron-like cell model with a GAP-43 reporter system. Such a model would have potential use in screening for novel therapeutics that upregulate GAP-43 and in the optimisation of electrical stimulation treatment in combination with these therapies. The key aim of the work was to design and construct a Cas9 expressing plasmid bearing a gRNA that targets GAP-43 cleavage and a donor plasmid bearing a reporter GFP or Neo cassette flanked with 5’ and 3’ GAP-43 homology arms (HAs) to facilitate the insertion of the reporter immediately 3’ of the GAP-43 promoter via CRISPR/Cas9 homology directed repair (HDR). Such an insertion would enable quantitative measurement of endogenous expression of the GAP-43 gene. Methods and Results: To guide the Cas9 nuclease to the target location, GAP-43 gRNA oligomers were designed and cloned downstream of the U6 promoter in the Cas9 expression plasmid px330, which also expresses the Cas9 gene and the cloned gRNA when transfected into cells. For CRISPR/Cas9 HDR, the 5’ and 3’ GAP-43 HAs were amplified from mouse genomic DNA and cloned into the donor plasmid using Gibson Assembly so that they flanked the reporter cassette. Results: This work successfully constructed the Cas9 gRNA expressing plasmid to target cleavage of the GAP-43 gene in mouse cell lines and has provided the complete design and construction foundation for generation of the reporter system for the endogenous GAP-43 gene in mice.

The use of CRISPR-Cas9 technology for targeted gene therapy in Parkinson's disease focusing on LRRK2 and SNCA genes

Parkinson's disease (PD) is a neurodegenerative disorder characterized by the progressive degeneration of dopaminergic neurons in the substantia nigra pars compacta, leading to the start of motor and non-motor symptoms. These include bradykinesia, resting tremor, rigidity, and postural instability, alongside cognitive impairments, mood disorders, and autonomic dysfunctions. The hallmark of PD is the accumulation of α-synuclein protein aggregates, forming Lewy bodies within the neurons. Given the numerous causes of Parkinson’s, involving genetic, epigenetic, and environmental factors, therapeutic treatments are hard to come by. Traditional pharmacological treatments, such as levodopa and dopamine agonists, primarily offer symptomatic relief and are associated with diminishing efficacy over time. Therefore, there is a need for new therapeutic approaches that target the disease at its roots. CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) technology, with the RNA-guided Cas9 endonuclease enables precise, targeted modifications at the genomic level. This technology offers potential for terminating the genetic factors causing parkinsons and aid in developing gene-based therapies. By attempting to regulate gene expression, CRISPR holds promise for addressing the fundamental causes of neurodegeneration in Parkinsons. One of the key genetic targets in PD is the leucine-rich repeat kinase 2 (LRRK2) gene, where specific gain-of- function mutations, such as G2019S, lead to hyperactivation of its kinase activity, contributing to neurotoxicity. CRISPR-Cas9 can be employed to introduce precise double-strand breaks at the mutation site, followed by homology-directed repair (HDR) to restore the wild-type sequence, thereby normalizing LRRK2 function and preventing neuronal death. Similarly, CRISPR technology can target the SNCA gene, which encodes α-synuclein. Pathogenic duplicates or triplicates of SNCA result in overproduction of α-synuclein and subsequent aggregation. Utilizing CRISPR to disrupt these extra copies can reduce α-synuclein levels, alleviating its toxic effects.CRISPR activation (CRISPRa) and interference (CRISPRi) systems enable fine-tuned upregulation or downregulation of gene expression, offering strategies to increase the expression of neuroprotective factors or suppress the expression of deleterious genes.

Gene targeting in Arabidopsis through one-armed homology-directed repair.

Gene targeting in Arabidopsis through one-armed homology-directed repair.

Comparison of Gene-Editing Approaches for Severe Congenital Neutropenia-Causing Mutations in the ELANE Gene.

Safety considerations for gene therapies of inherited preleukemia syndromes, including severe congenital neutropenia (CN), are paramount. We compared several strategies for CRISPR/Cas9 gene editing of autosomal-dominant ELANE mutations in CD34+ cells from two CN patients head-to-head. We tested universal and allele-specific ELANE knockout, ELANE mutation correction by homology-directed repair (HDR) with AAV6, and allele-specific HDR with ssODN. All strategies were not toxic, had at least 30% editing, and rescued granulopoiesis in vitro. In contrast to published data, allele-specific indels in the last exon of ELANE also restored granulopoiesis. Moreover, by implementing patient-derived induced pluripotent stem cells for GUIDE-Seq off-target analysis, we established a clinically relevant "personalized" assessment of off-target activity of gene editing on the background of the patient's genome. We found that allele-specific approaches had the most favorable off-target profiles. Taken together, a well-defined head-to-head comparison pipeline for selecting the appropriate gene therapy is essential for diseases, with several gene editing strategies available.

Next-generation cell-penetrating antibodies for tumor targeting and RAD51 inhibition.

Monoclonal antibody therapies for cancer have demonstrated extraordinary clinical success in recent years. However, these strategies are thus far mostly limited to specific cell surface antigens, even though many disease targets are found intracellularly. Here we report studies on the humanization of a full-length, nucleic acid binding, monoclonal lupus-derived autoantibody, 3E10, which exhibits a novel mechanism of cell penetration and tumor specific targeting. Comparing humanized variants of 3E10, we demonstrate that cell uptake depends on the nucleoside transporter ENT2, and that faster cell uptake and superior in vivo tumor targeting are associated with higher affinity nucleic acid binding. We show that one human variant retains the ability of the parental 3E10 to bind RAD51, serving as a synthetically lethal inhibitor of homology-directed repair in vitro. These results provide the basis for the rational design of a novel antibody platform for therapeutic tumor targeting with high specificity following systemic administration.

AAV-mediated gene therapies by miniature gene editing tools.

The advent of CRISPR-Cas has revolutionized precise gene editing. While pioneering CRISPR nucleases like Cas9 and Cas12 generate targeted DNA double-strand breaks (DSB) for knockout or homology-directed repair, next generation CRISPR technologies enable gene editing without DNA DSB. Base editors directly convert bases, prime editors make diverse alterations, and dead Cas-regulator fusions allow nuanced control of gene expression, avoiding potentially risks like translocations. Meanwhile, the discovery of diminutive Cas12 orthologs and Obligate Mobile Element-Guided Activity (OMEGA) nucleases has overcome cargo limitations of adeno-associated viral vectors, expanding prospects for in vivo therapeutic delivery. Here, we review the ever-evolving landscape of cutting-edge gene editing tools, focusing on miniature Cas12 orthologs and OMEGA effectors amenable to single AAV packaging. We also summarize CRISPR therapies delivered using AAV vectors, discuss challenges such as efficiency and specificity, and look to the future of this transformative field of in vivo gene editing enabled by AAV vectors delivery.

BRCA2 C-terminal clamp restructures RAD51 dimers to bind B-DNA for replication fork stability.

Tumor suppressor protein BRCA2 acts with RAD51 in replication-fork protection (FP) and homology-directed DNA break repair (HDR). Critical for cancer etiology and therapy resistance, BRCA2 C-terminus was thought to stabilize RAD51-filaments after they assemble on single-stranded (ss)DNA. Here we determined the detailed crystal structure for BRCA2 C-terminal interaction-domain (TR2i) with ATP-bound RAD51 prior to DNA binding. In contrast to recombinogenic RAD51-filaments comprising extended ATP-bound RAD51 dimers, TR2i unexpectedly reshapes ATP-RAD51 into a unique dimer conformation accommodating double-stranded B-DNA binding unsuited for HDR initiation. Structural, biochemical, and molecular results with interface-guided mutations uncover TR2i's FP mechanism. Proline-driven secondary-structure stabilizes residue triads and spans the RAD51 dimer engaging pivotal interactions of RAD51 M210 and BRCA2 S3291/P3292, the cyclin-dependent kinase (CDK) phosphorylation site that toggles between FP during S-phase and HDR in G2. TR2i evidently acts as an allosteric clamp switching RAD51 from ssDNA to double-stranded and B-DNA binding enforcing FP over HDR.

FecBB mutation enhances follicle stimulating hormone sensitivity of granulosa cells by up-regulating the SMAD1/5–USF1–FSHR signaling pathway

The FecBB mutation, a single-point mutation (c.A746G; p.Q249R) in bone morphogenetic protein receptor type 1 B (BMPR1B), is associated with increased ovulation quotas and litter size in sheep. However, the regulatory mechanism of the FecBB mutation in increased fecundity remains to be elucidated. Therefore, creating an immortal cell model harboring the FecBB mutation would elucidate the regulatory mechanism of this mutation. Here, we report the creation of a human granulosa cell, COV434, model containing a homozygous FecBB mutation through homology-directed repair (HDR) induced by clustered, regularly-interspaced, short palindromic repeats–CRISPR-associated protein 9 along with a single-stranded oligodeoxynucleotide (ssODN) template. We found that the FecBB mutation enhanced the basal SMAD1/5 signaling activity in COV434 cells, leading to increased expression of FSHR, probably through increased formation of the SMAD1/5–SMAD4 complex to bind to the SBE element, which in turn promotes the binding of USF1 to the regulatory element E-box in the promoter of FSHR. Furthermore, the FecBB mutation substantially enhanced estradiol (E2) synthesis in granulosa cells under follicle stimulating hormone (FSH) stimulation, indicating an enhanced sensitivity to FSH, which may promote the growth of more small follicles into mature follicles, leading to increased fecundity. Our study provides novel insights into the possible regulatory mechanisms of FecBB mutations in increased fecundity.

Generation of SOX2ZsGreen reporter authentic porcine induced pluripotent stem cells

The transcription factor SOX2 plays a crucial role in pluripotency during embryogenesis. In this study, we successfully generated porcine induced pluripotent stem cells (piPSC) by transducing porcine fetal fibroblasts with doxycycline-inducible reprogramming lentiviral vectors. To enhance the utility of these piPSCs, we used a CRISPR/Cas9-based homology-directed repair (HDR) system to introduce a nls-zsGreen reporter in-frame and before the stop codon of the SOX2 coding sequence. The incorporation of the zsGreen reporter offers a novel opportunity for real-time monitoring of SOX2 expression and aiding in the characterization of piPSC lines.