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PLoS Biology May 2024The CRISPR-associated endonuclease Cas12a has become a powerful genome-editing tool in biomedical research due to its ease of use and low off-targeting. However, the...
The CRISPR-associated endonuclease Cas12a has become a powerful genome-editing tool in biomedical research due to its ease of use and low off-targeting. However, the size of Cas12a severely limits clinical applications such as adeno-associated virus (AAV)-based gene therapy. Here, we characterized a novel compact Cas12a ortholog, termed EbCas12a, from the metagenome-assembled genome of a currently unclassified Erysipelotrichia. It has the PAM sequence of 5'-TTTV-3' (V = A, G, C) and the smallest size of approximately 3.47 kb among the Cas12a orthologs reported so far. In addition, enhanced EbCas12a (enEbCas12a) was also designed to have comparable editing efficiency with higher specificity to AsCas12a and LbCas12a in mammalian cells at multiple target sites. Based on the compact enEbCas12a, an all-in-one AAV delivery system with crRNA for Cas12a was developed for both in vitro and in vivo applications. Overall, the novel smallest high-fidelity enEbCas12a, this first case of the all-in-one AAV delivery for Cas12a could greatly boost future gene therapy and scientific research.
Topics: Dependovirus; Humans; Gene Editing; Genetic Vectors; CRISPR-Cas Systems; Animals; HEK293 Cells; Genetic Therapy; CRISPR-Associated Proteins; Mice; Endodeoxyribonucleases; Bacterial Proteins
PubMed: 38814985
DOI: 10.1371/journal.pbio.3002619 -
Nature Communications May 2024The mammalian SWI/SNF-like BAF complexes play critical roles during animal development and pathological conditions. Previous gene deletion studies and characterization...
The mammalian SWI/SNF-like BAF complexes play critical roles during animal development and pathological conditions. Previous gene deletion studies and characterization of human gene mutations implicate that the complexes both repress and activate a large number of genes. However, the direct function of the complexes in cells remains largely unclear due to the relatively long-term nature of gene deletion or natural mutation. Here we generate a mouse line by knocking in the auxin-inducible degron tag (AID) to the Smarca4 gene, which encodes BRG1, the essential ATPase subunit of the BAF complexes. We show that the tagged BRG1 can be efficiently depleted by osTIR1 expression and auxin treatment for 6 to 10 h in CD4 + T cells, hepatocytes, and fibroblasts isolated from the knock-in mice. The acute depletion of BRG1 leads to decreases in nascent RNAs and RNA polymerase II binding at a large number of genes, which are positively correlated with the loss of BRG1. Further, these changes are correlated with diminished accessibility at DNase I Hypersensitive Sites (DHSs) and p300 binding. The acute BRG1 depletion results in three major patterns of nucleosome shifts leading to narrower nucleosome spacing surrounding transcription factor motifs and at enhancers and transcription start sites (TSSs), which are correlated with loss of BRG1, decreased chromatin accessibility and decreased nascent RNAs. Acute depletion of BRG1 severely compromises the Trichostatin A (TSA) -induced histone acetylation, suggesting a substantial interplay between the chromatin remodeling activity of BRG1 and histone acetylation. Our data suggest BRG1 mainly plays a direct positive role in chromatin accessibility, RNAPII binding, and nascent RNA production by regulating nucleosome positioning and facilitating transcription factor binding to their target sites.
Topics: Animals; Transcription Factors; DNA Helicases; Nuclear Proteins; Mice; Nucleosomes; Indoleacetic Acids; RNA Polymerase II; Fibroblasts; Gene Knock-In Techniques; Hepatocytes; E1A-Associated p300 Protein; Transcriptional Activation; Transcription, Genetic; Histones; Deoxyribonuclease I; Chromatin; Humans
PubMed: 38811575
DOI: 10.1038/s41467-024-48911-z -
International Journal of Molecular... May 2024The myostatin () gene also regulates the developmental balance of skeletal muscle after birth, and has long been linked to age-related muscle wasting. Many rodent...
The myostatin () gene also regulates the developmental balance of skeletal muscle after birth, and has long been linked to age-related muscle wasting. Many rodent studies have shown a correlation between and age-related diseases. It is unclear how and age-associated muscle loss in other animals are related. In this study, we utilized gene-edited bovine skeletal muscle cells to investigate the mechanisms relating to and muscle cell senescence. The expression of was higher in older individuals than in younger individuals. We obtained consecutively passaged senescent cells and performed senescence index assays and transcriptome sequencing. We found that senescence hallmarks and the senescence-associated secretory phenotype (SASP) were decreased in long-term-cultured myostatin inactivated (MT-KO) bovine skeletal muscle cells (bSMCs). Using cell signaling profiling, was shown to regulate the SASP, predominantly through the cycle GMP-AMP synthase-stimulator of antiviral genes (cGAS-STING) pathway. An in-depth investigation by chromatin immunoprecipitation (ChIP) analysis revealed that influenced three prime repair exonuclease 1 () expression through the SMAD2/3 complex. The downregulation of contributed to the activation of the MSTN-SMAD2/3-TREX1 signaling axis, influencing the secretion of SASP, and consequently delaying the senescence of bSMCs. This study provided valuable new insight into the role of in cell senescence in large animals.
Topics: Animals; Myostatin; Cattle; Cellular Senescence; Exodeoxyribonucleases; Signal Transduction; Muscle Fibers, Skeletal; Muscle, Skeletal; Phosphoproteins; Cells, Cultured
PubMed: 38791317
DOI: 10.3390/ijms25105277 -
Sheng Wu Gong Cheng Xue Bao = Chinese... May 2024Programmable nucleases-based genome editing systems offer several advantages, such as high editing efficiency, high product purity, and fewer editing by-products. They... (Review)
Review
Programmable nucleases-based genome editing systems offer several advantages, such as high editing efficiency, high product purity, and fewer editing by-products. They have been widely used in biopharmaceutical research and crop engineering. Given the diverse needs for research and application, developing functional base editors has become a major focus in the field of genome editing. Currently, genome editing systems derived from clustered regularly interspaced short palindromic repeats and CRISPR-associated (CRISPR-Cas) and transcription activator-like effector (TALE) systems include single base editors, dual base editors, mitochondrial base editors, and CRISPR-related transposase systems. This review provides a comprehensive overview of the development of base editing systems, summarizes the characteristics, off-target effects, optimization, and improvement strategies of various base editors, and provides insights for further improvement and application of genome editing systems.
Topics: Gene Editing; CRISPR-Cas Systems; Transcription Activator-Like Effector Nucleases; Clustered Regularly Interspaced Short Palindromic Repeats; Genetic Engineering; Humans
PubMed: 38783797
DOI: 10.13345/j.cjb.230615 -
BMC Biology May 2024Many efforts have been made to improve the precision of Cas9-mediated gene editing through increasing knock-in efficiency and decreasing byproducts, which proved to be...
BACKGROUND
Many efforts have been made to improve the precision of Cas9-mediated gene editing through increasing knock-in efficiency and decreasing byproducts, which proved to be challenging.
RESULTS
Here, we have developed a human exonuclease 1-based genome-editing tool, referred to as exonuclease editor. When compared to Cas9, the exonuclease editor gave rise to increased HDR efficiency, reduced NHEJ repair frequency, and significantly elevated HDR/indel ratio. Robust gene editing precision of exonuclease editor was even superior to the fusion of Cas9 with E1B or DN1S, two previously reported precision-enhancing domains. Notably, exonuclease editor inhibited NHEJ at double strand breaks locally rather than globally, reducing indel frequency without compromising genome integrity. The replacement of Cas9 with single-strand DNA break-creating Cas9 nickase further increased the HDR/indel ratio by 453-fold than the original Cas9. In addition, exonuclease editor resulted in high microhomology-mediated end joining efficiency, allowing accurate and flexible deletion of targeted sequences with extended lengths with the aid of paired sgRNAs. Exonuclease editor was further used for correction of DMD patient-derived induced pluripotent stem cells, where 30.0% of colonies were repaired by HDR versus 11.1% in the control.
CONCLUSIONS
Therefore, the exonuclease editor system provides a versatile and safe genome editing tool with high precision and holds promise for therapeutic gene correction.
Topics: Gene Editing; Humans; Exodeoxyribonucleases; CRISPR-Cas Systems; HEK293 Cells; DNA Repair Enzymes
PubMed: 38769511
DOI: 10.1186/s12915-024-01918-w -
Journal of Nanobiotechnology May 2024The detection of carcinoembryonic antigen (CEA) holds significant importance in the early diagnosis of cancer. However, current methods are hindered by limited...
The detection of carcinoembryonic antigen (CEA) holds significant importance in the early diagnosis of cancer. However, current methods are hindered by limited accessibility and specificity. This study proposes a rapid and convenient Cas12a-based assay for the direct detection of CEA in clinical serum samples, aiming to address these limitations. The protocol involves a rolling machine operation, followed by a 5-min Cas12a-mediated cleavage process. The assay demonstrates the capability to detect human serum with high anti-interference performance and a detection limit as low as 0.2 ng/mL. The entire testing procedure can be accomplished in 75 min without centrifugation steps, and successfully reduced the limit of detection of traditional DNA walking machine by 50 folds. Overall, the testing procedure can be easily implemented in clinical settings.
Topics: Carcinoembryonic Antigen; Humans; Biosensing Techniques; Limit of Detection; DNA; CRISPR-Cas Systems; Endodeoxyribonucleases; Nucleic Acid Amplification Techniques; CRISPR-Associated Proteins; Bacterial Proteins
PubMed: 38762451
DOI: 10.1186/s12951-024-02535-z -
Cell Reports May 2024Phagocytic macrophages are crucial for innate immunity and tissue homeostasis. Most tissue-resident macrophages develop from embryonic precursors that populate every...
Phagocytic macrophages are crucial for innate immunity and tissue homeostasis. Most tissue-resident macrophages develop from embryonic precursors that populate every organ before birth to lifelong self-renew. However, the mechanisms for versatile macrophage differentiation remain unknown. Here, we use in vivo genetic and cell biological analysis of the Drosophila larval hematopoietic organ, the lymph gland that produces macrophages. We show that the developmentally regulated transient activation of caspase-activated DNase (CAD)-mediated DNA strand breaks in intermediate progenitors is essential for macrophage differentiation. Insulin receptor-mediated PI3K/Akt signaling regulates the apoptosis signal-regulating kinase 1 (Ask1)/c-Jun kinase (JNK) axis to control sublethal levels of caspase activation, causing DNA strand breaks during macrophage development. Furthermore, caspase activity is also required for embryonic-origin macrophage development and efficient phagocytosis. Our study provides insights into developmental signaling and CAD-mediated DNA strand breaks associated with multifunctional and heterogeneous macrophage differentiation.
Topics: Animals; Macrophages; Cell Differentiation; DNA Damage; Phagocytosis; Drosophila Proteins; Signal Transduction; Caspases; Enzyme Activation; Deoxyribonucleases; Drosophila melanogaster; Proto-Oncogene Proteins c-akt; Phosphatidylinositol 3-Kinases
PubMed: 38761374
DOI: 10.1016/j.celrep.2024.114251 -
Nature Communications May 2024Intestinal homeostasis is maintained by the response of gut-associated lymphoid tissue to bacteria transported across the follicle associated epithelium into the...
Intestinal homeostasis is maintained by the response of gut-associated lymphoid tissue to bacteria transported across the follicle associated epithelium into the subepithelial dome. The initial response to antigens and how bacteria are handled is incompletely understood. By iterative application of spatial transcriptomics and multiplexed single-cell technologies, we identify that the double negative 2 subset of B cells, previously associated with autoimmune diseases, is present in the subepithelial dome in health. We show that in this location double negative 2 B cells interact with dendritic cells co-expressing the lupus autoantigens DNASE1L3 and C1q and microbicides. We observe that in humans, but not in mice, dendritic cells expressing DNASE1L3 are associated with sampled bacteria but not DNA derived from apoptotic cells. We propose that fundamental features of autoimmune diseases are microbiota-associated, interacting components of normal intestinal immunity.
Topics: Animals; Humans; Mice; B-Lymphocytes; Gastrointestinal Microbiome; Endodeoxyribonucleases; Dendritic Cells; Lymphoid Tissue; Female; Mice, Inbred C57BL; Intestinal Mucosa; Male
PubMed: 38744839
DOI: 10.1038/s41467-024-48267-4 -
Cell Death & Disease May 2024Pirh2 is an E3 ubiquitin ligase known to regulate the DNA damage responses through ubiquitylation of various participating signaling factors. DNA damage is a key...
Pirh2 is an E3 ubiquitin ligase known to regulate the DNA damage responses through ubiquitylation of various participating signaling factors. DNA damage is a key pathological contributor to Alzheimer's disease (AD), therefore, the role of Pirh2 was investigated in streptozotocin and oligomer Aβ induced rodent experimental model of AD. Pirh2 protein abundance increased during AD conditions, and transient silencing of Pirh2 inhibited the disease-specific pathological markers like level of p-Tau, βamyloid, acetylcholinesterase activity, and neuronal death. Biochemically, Pirh2 silencing significantly attenuated the oxidative stress, depleted mitochondrial membrane potential, cytochrome c translocation from mitochondria to cytosol, and depleted mitochondrial complex-I activity, and ATP level. Pirh2 silencing also inhibited the altered level of VDAC1, hsp75, hexokinase1, t-Bid, caspase-9, and altered level of apoptotic proteins (Bcl-2, Bax). MALDI-TOF/TOF, co-immunoprecipitation, and UbcH13-linked ubiquitylation assay confirmed the interaction of Pirh2 with cytochrome c and the role of Pirh2 in ubiquitylation of cytochrome c, along with Pirh2-dependent altered proteasome activity. Additionally, Pirh2 silencing further inhibited the translocation of mitochondrion-specific endonuclease G and apoptosis-inducing factors to the nucleus and DNA damage. In conclusion, findings suggested the significant implication of Pirh2 in disease pathogenesis, particularly through impaired mitochondrial function, including biochemical alterations, translocation of cytochrome c, endonuclease G and apoptosis-inducing factor, DNA damage, and neuronal apoptosis.
Topics: Alzheimer Disease; Animals; Cytochromes c; Mitochondria; Neurons; Oxidative Stress; Rats; Male; Ubiquitin-Protein Ligases; Amyloid beta-Peptides; Membrane Potential, Mitochondrial; Ubiquitination; Humans; Apoptosis; Cell Death; Rats, Sprague-Dawley; Disease Models, Animal; Endodeoxyribonucleases
PubMed: 38740775
DOI: 10.1038/s41419-024-06662-1 -
Nature Communications May 2024The long interspersed nuclear element-1 (LINE-1 or L1) retrotransposon is the only active autonomously replicating retrotransposon in the human genome. L1 harms the cell...
The long interspersed nuclear element-1 (LINE-1 or L1) retrotransposon is the only active autonomously replicating retrotransposon in the human genome. L1 harms the cell by inserting new copies, generating DNA damage, and triggering inflammation. Therefore, L1 inhibition could be used to treat many diseases associated with these processes. Previous research has focused on inhibition of the L1 reverse transcriptase due to the prevalence of well-characterized inhibitors of related viral enzymes. Here we present the L1 endonuclease as another target for reducing L1 activity. We characterize structurally diverse small molecule endonuclease inhibitors using computational, biochemical, and biophysical methods. We also show that these inhibitors reduce L1 retrotransposition, L1-induced DNA damage, and inflammation reinforced by L1 in senescent cells. These inhibitors could be used for further pharmacological development and as tools to better understand the life cycle of this element and its impact on disease processes.
Topics: Humans; Long Interspersed Nucleotide Elements; Endonucleases; Enzyme Inhibitors; DNA Damage; Small Molecule Libraries; Cellular Senescence; Deoxyribonuclease I
PubMed: 38719805
DOI: 10.1038/s41467-024-48066-x