RESUMO
Most insertions or deletions generated by CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9) endonucleases are short (<25 bp), but unpredictable on-target long DNA deletions (>500 bp) can be observed. The possibility of generating long on-target DNA deletions poses safety risks to somatic genome editing and makes the outcomes of genome editing less predictable. Methods for generating refined mutations are desirable but currently unavailable. Here, we show that fusing Escherichia coli DNA polymerase I or the Klenow fragment to Cas9 greatly increases the frequencies of 1-bp deletions and decreases >1-bp deletions or insertions. Importantly, doing so also greatly decreases the generation of long deletions, including those >2 kb. In addition, templated insertions (the insertion of the nucleotide 4 nt upstream of the protospacer adjacent motif) were increased relative to other insertions. Counteracting DNA resection was one of the mechanisms perturbing deletion sizes. Targeting DNA polymerase to double-strand breaks did not increase off-targets or base substitution rates around the cleavage sites, yet increased editing efficiency in primary cells. Our strategy makes it possible to generate refined DNA mutations for improved safety without sacrificing efficiency of genome editing.
Assuntos
Sistemas CRISPR-Cas , Edição de Genes , Proteína 9 Associada à CRISPR/genética , Proteína 9 Associada à CRISPR/metabolismo , DNA/genética , DNA/metabolismo , DNA Polimerase Dirigida por DNA/metabolismo , Edição de Genes/métodosRESUMO
The designer nucleases, including Zinc Finger Nuclease (ZFN), Transcription Activator-Like Effector Nuclease (TALEN), and Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated (CRISPR/Cas), have been widely used for mechanistic studies, animal model generation, and gene therapy development. Clinical trials using designer nucleases to treat genetic diseases or cancers are showing promising results. Despite rapid progress, potential off-targets and host immune responses are challenges to be addressed for in vivo uses, especially in clinical applications. Short-term expression of the designer nucleases is necessary to reduce both risks. Currently, delivery methods enabling transient expression of designer nucleases are being pursued. Among these, virus-like particles as delivery vehicles for short-term designer nuclease expression have received much attention. This review will summarize recent developments in using virus-like particles (VLPs) for safe delivery of gene editing effectors to complement our last review on the same topic. First, we introduce some background information on how VLPs can be used for safe and efficient CRISPR/Cas9 delivery. Then, we summarize recently developed virus-like particles as genome editing vehicles. Finally, we discuss applications and future directions.
Assuntos
Sistemas CRISPR-Cas , Edição de Genes , Animais , Sistemas CRISPR-Cas/genética , Endonucleases/metabolismo , Eucariotos/genética , Edição de Genes/métodos , Nucleases de Dedos de Zinco/genéticaRESUMO
Transient expression of the CRISPR/Cas9 machinery will not only reduce risks of mutagenesis from off-target activities, but also decrease possible immune response to Cas9 protein. Building on our recent developing of a system able to package up to 100 copies of Cas9 mRNA in each lentivirus-like particle (LVLP) via the specific interaction between aptamer and aptamer-binding proteins (ABP), here we develop a lentiviral capsid-based bionanoparticle system, which allows efficient packaging of Cas9/sgRNA ribonucleoprotein (RNP). We show that replacing the Tetraloop of sgRNA scaffold with a com aptamer preserves the functions of the guide RNA, and the com-modified sgRNA can package Cas9/sgRNA RNP into lentivirus-like particles via the specific interactions between ABP and aptamer, and sgRNA and Cas9 protein. These RNP bionanoparticles generated Indels on different targets in different cells with efficiencies similar to or better than our recently described Cas9 mRNA LVLPs. The new system showed fast action and reduced off-target rates, and makes it more convenient and efficient in delivering Cas9 RNPs for transient Cas9 expression and efficient genome editing.
Assuntos
Proteína 9 Associada à CRISPR/genética , Sistemas CRISPR-Cas/genética , Edição de Genes/métodos , Técnicas de Transferência de Genes , Ribonucleoproteínas/genética , Aptâmeros de Nucleotídeos/química , Aptâmeros de Nucleotídeos/genética , Capsídeo/metabolismo , Proteínas do Capsídeo/genética , Vetores Genéticos/genética , Células HEK293 , Humanos , Lentivirus/genética , Nanopartículas/química , Nanopartículas/metabolismoRESUMO
The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) system discovered using bacteria has been repurposed for genome editing in human cells. Transient expression of the editor proteins (e.g. Cas9 protein) is desirable to reduce the risk of mutagenesis from off-target activity. Using the specific interaction between bacteriophage RNA-binding proteins and their RNA aptamers, we developed a system able to package up to 100 copies of Staphylococcus aureus Cas9 (SaCas9) mRNA in each lentivirus-like bionanoparticle (LVLP). The SaCas9 LVLPs mediated transient SaCas9 expression and achieved highly efficient genome editing in the presence of guide RNA. Lower off-target rates occurred in cells transduced with LVLPs containing SaCas9 mRNA, compared with cells transduced with adeno-associated virus or lentivirus expressing SaCas9. Our LVLP system may be useful for efficiently delivering Cas9 mRNA to cell lines and primary cells for in vitro and in vivo gene editing applications.
Assuntos
Proteínas de Bactérias/genética , Proteína 9 Associada à CRISPR/genética , Sistemas CRISPR-Cas , Edição de Genes/métodos , Nanopartículas/metabolismo , Transfecção/métodos , Proteínas de Bactérias/metabolismo , Sequência de Bases , Proteína 9 Associada à CRISPR/metabolismo , Dependovirus/genética , Dependovirus/metabolismo , Genes Reporter , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Lentivirus/genética , Lentivirus/metabolismo , Nanopartículas/química , Plasmídeos/química , Plasmídeos/metabolismo , RNA Guia de Cinetoplastídeos/genética , RNA Guia de Cinetoplastídeos/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Alinhamento de Sequência , Staphylococcus aureus/química , Staphylococcus aureus/enzimologiaRESUMO
Gene conversion is a process of transferring genetic material from one homologous sequence to another. Most reported gene conversions are meiotic although mitotic gene conversion is also described. When using CRISPR/Cas9 to target the human hemoglobin subunit beta (HBB) gene, hemoglobin subunit delta (HBD) gene footprints were observed in HBB gene. However, it is unclear whether these were the results of gene conversion or PCR-mediated sequence shuffling between highly homologous sequences. Here we provide evidence that the HBD footprints in HBB were indeed results of gene conversion. We demonstrated that the CRISPR/Cas9 facilitated unidirectional sequence transfer from the homologous gene without double-strand breaks (DSB) to the one with DSBs, and showed that the rates of HBD footprint in HBB were positively correlated to the HBB insertion and deletion rates. We further showed that when targeting HBD gene, HBB footprints could also be observed in HBD gene. The mitotic gene conversion was observed not only in immortalized HEK293T cells, but also in human primary cells. Our work reveals mitotic gene conversion as an often overlooked effect of CRISPR/Cas9-mediated genome editing.
Assuntos
Sistemas CRISPR-Cas , Conversão Gênica , Edição de Genes , Células HEK293 , HumanosRESUMO
Previous studies have confirmed that spermatogenesis in homozygous Immp2l mutant male mice was normal at the age of 6 months, but was significantly abnormal at the age of 13 months. Meanwhile, oxidative stress is reported to be involved in spermatogenic impairment in old mutant mice. However, it is unclear whether antioxidant treatment is a suitable intervention for improving spermatogenesis in old mutant mice. This study sought to investigate the effect of mitochondria-targeted antioxidant SkQ1 on spermatogenesis in homozygous Immp2l mutant mice. Immp2l mutant mice were treated with the mitochondria-targeted antioxidant SkQ1 from the age of 6 weeks until 13 months. SkQ1 treatment significantly improved spermatogenesis in old Immp2 l mutant mice. Moreover, SkQ1 treatment improved the morphology of testicular seminiferous tubules, significantly reduced the apoptosis of germ cells and increased the level of GPX4 expression in old Immp2 l mutant mice. In conclusion, our data suggest that the mitochondria-targeted antioxidant SkQ1 is effective in improving spermatogenesis in Immp2 l mutant mice and might be used for the treatment of male infertility.
Assuntos
Antioxidantes/farmacologia , Endopeptidases/genética , Infertilidade Masculina/tratamento farmacológico , Mitocôndrias/efeitos dos fármacos , Proteínas Mitocondriais/genética , Plastoquinona/análogos & derivados , Espermatogênese/efeitos dos fármacos , Animais , Apoptose/efeitos dos fármacos , Modelos Animais de Doenças , Homozigoto , Humanos , Infertilidade Masculina/genética , Masculino , Camundongos , Camundongos Transgênicos , Mitocôndrias/genética , Mitocôndrias/patologia , Mutação , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/genética , Plastoquinona/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Túbulos Seminíferos/efeitos dos fármacos , Túbulos Seminíferos/patologia , Espermatogênese/genética , Espermatozoides/efeitos dos fármacos , Espermatozoides/fisiologiaRESUMO
Currently, the human MEX3C gene is known to encode an RNA-binding protein of 659 amino acid residues. Here we show that the MEX3C gene has alternative splicing forms giving rise to multiple MEX3C variants, and some cells express MEX3C transcripts coding for short MEX3C isoforms but not transcripts for MEX3C(659AA) MEX3C(659AA) functions as an adaptor protein for Exportin 1 (XPO1)-mediated nuclear export since it increases the cytoplasmic distribution of poly(A)(+) RNA and since addition of the nuclear export signal (NES) sequence to a short MEX3C isoform MEX3C(464AA) confers similar cytoplasmic poly(A)(+) RNA accumulation activity as MEX3C(659AA) FOS mRNA is a potential MEX3C target mRNA. One mechanism by which MEX3C(659AA) could regulate FOS mRNA is by promoting its nuclear export. Overexpressing MEX3C(659AA) significantly increased FOS mRNA expression, whereas mutating the NES of MEX3C(659AA) and treating cells with leptomycin B to inhibit XPO1-mediated nuclear export attenuated FOS upregulation. FOS mRNA is unstable in somatic cells but less so in oocytes; how it is stabilized in the oocytes is unknown. Transcripts for the mouse counterpart of human MEX3C(659AA) (MEX3C(652AA)) are specifically expressed in developing oocytes in the ovary, although total Mex3c transcripts are expressed in both granulosa cells and oocytes. The specific expression of this long MEX3C isoform in oocytes and its ability to enhance FOS mRNA nuclear export and stability all suggest that MEX3C(659AA) is an RNA-binding protein that preserves maternal FOS mRNA in oocytes.
Assuntos
Genes fos , Oócitos/metabolismo , Ovário/metabolismo , RNA Mensageiro Estocado/genética , Proteínas de Ligação a RNA/genética , Transporte Ativo do Núcleo Celular , Animais , Feminino , Regulação da Expressão Gênica , Células HEK293 , Humanos , Camundongos , Camundongos Transgênicos , Oogênese/genética , Especificidade de Órgãos/genética , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas Proto-Oncogênicas c-fos/metabolismo , RNA Mensageiro Estocado/metabolismo , Proteínas de Ligação a RNA/metabolismoRESUMO
Mutations in Cl-/H+ antiporter ClC-5 cause Dent's disease type 1 (DD1), a rare tubulopathy that progresses to renal fibrosis and kidney failure. Here, we have used DD1 human cellular models and renal tissue from DD1 mice to unravel the role of ClC-5 in renal fibrosis. Our results in cell systems have shown that ClC-5 deletion causes an increase in collagen I (Col I) and IV (Col IV) intracellular levels by promoting their transcription through the ß-catenin pathway and impairing their lysosomal-mediated degradation. Increased production of Col I/IV in ClC-5-depleted cells ends up in higher release to the extracellular medium, which may lead to renal fibrosis. Furthermore, our data have revealed that 3-mo-old mice lacking ClC-5 (Clcn5 +/- and Clcn5 -/- ) present higher renal collagen deposition and fibrosis than WT mice. Altogether, we describe a new regulatory mechanism for collagens' production and release by ClC-5, which is altered in DD1 and provides a better understanding of disease progression to renal fibrosis.
Assuntos
Canais de Cloreto , Fibrose , Lisossomos , Camundongos Knockout , beta Catenina , Animais , Canais de Cloreto/metabolismo , Canais de Cloreto/genética , Lisossomos/metabolismo , Humanos , Camundongos , beta Catenina/metabolismo , Fibrose/metabolismo , Rim/metabolismo , Rim/patologia , Colágeno Tipo I/metabolismo , Doença de Dent/metabolismo , Doença de Dent/genética , Proteólise , Transdução de SinaisRESUMO
PURPOSE: Chordoma is a rare and aggressive bone cancer driven by the developmental transcription factor brachyury. Efforts to target brachyury are hampered by the absence of ligand-accessible small-molecule binding pockets. Genome editing with CRISPR systems provides an unprecedented opportunity to modulate undruggable transcription factor targets. However, delivery of CRISPR remains a bottleneck for in vivo therapy development. The aim was to investigate the in vivo therapeutic efficiency of Cas9/guide RNA (gRNA) ribonucleoprotein (RNP) delivery through a novel virus-like particle (VLP) by fusing an aptamer-binding protein to the lentiviral nucleocapsid protein. METHODS: The p24 based ELISA and transmission electron microscopy were used to determine the characterization of engineered VLP-packaged Cas9/gRNA RNP. The deletion efficiency of brachyury gene in chordoma cells and tissues was measured by genome cleavage detection assay. RT-PCR, Western blot, immunofluorescence staining, and IHC were employed to test the function of brachyury deletion. Cell growth and tumor volume were measured to evaluate the therapeutic efficiency of brachyury deletion by VLP-packaged Cas9/gRNA RNP. RESULTS: Our "all-in-one" VLP-based Cas9/gRNA RNP system allows for transient expression of Cas9 in chordoma cells, but maintains efficient editing capacity leading to approximately 85% knockdown of brachyury with subsequent inhibition of chordoma cell proliferation and tumor progression. In addition, this VLP-packaged brachyury-targeting Cas9 RNP avoids systemic toxicities in vivo. CONCLUSION: Our preclinical studies demonstrate the potential of VLP-based Cas9/gRNA RNP gene therapy for the treatment of brachyury-dependent chordoma.
RESUMO
OBJECTIVE: We previously reported that mutations in inner mitochondrial membrane peptidase 2-like (Immp2l) increase infarct volume, enhance superoxide production, and suppress mitochondrial respiration after transient cerebral focal ischemia and reperfusion injury. The present study investigated the impact of heterozygous Immp2l mutation on mitochondria function after ischemia and reperfusion injury in mice. METHODS: Mice were subjected to middle cerebral artery occlusion for 1 h followed by 0, 1, 5, and 24 h of reperfusion. The effects of Immp2l+/- on mitochondrial membrane potential, mitochondrial respiratory complex III activity, caspase-3, and apoptosis-inducing factor (AIF) translocation were examined. RESULTS: Immp2l+/- increased ischemic brain damage and the number of TUNEL-positive cells compared with wild-type mice. Immp2l+/- led to mitochondrial damage, mitochondrial membrane potential depolarization, mitochondrial respiratory complex III activity suppression, caspase-3 activation, and AIF nuclear translocation. CONCLUSION: The adverse impact of Immp2l+/- on the brain after ischemia and reperfusion might be related to mitochondrial damage that involves depolarization of the mitochondrial membrane potential, inhibition of the mitochondrial respiratory complex III, and activation of mitochondria-mediated cell death pathways. These results suggest that patients with stroke carrying Immp2l+/- might have worse and more severe infarcts, followed by a worse prognosis than those without Immp2l mutations.
Assuntos
Ataque Isquêmico Transitório , Traumatismo por Reperfusão , Animais , Camundongos , Caspase 3/genética , Caspase 3/metabolismo , Complexo III da Cadeia de Transporte de Elétrons/metabolismo , Infarto da Artéria Cerebral Média/genética , Infarto da Artéria Cerebral Média/metabolismo , Ataque Isquêmico Transitório/metabolismo , Membranas Mitocondriais/metabolismo , Mutação , Traumatismo por Reperfusão/metabolismoRESUMO
Background: The liver metastasis accompanied with the loss of liver function is one of the most common complications in patients with triple-negative breast cancers (TNBC). Lineage reprogramming, as a technique direct inducing the functional cell types from one lineage to another lineage without passing through an intermediate pluripotent stage, is promising in changing cell fates and overcoming the limitations of primary cells. However, most reprogramming techniques are derived from human fibroblasts, and whether cancer cells can be reversed into hepatocytes remains elusive. Methods: Herein, we simplify preparation of reprogramming reagents by expressing six transcriptional factors (HNF4A, FOXA2, FOXA3, ATF5, PROX1, and HNF1) from two lentiviral vectors, each expressing three factors. Then the virus was transduced into MDA-MB-231 cells to generated human induced hepatocyte-like cells (hiHeps) and single-cell sequencing was used to analyze the fate for the cells after reprogramming. Furthermore, we constructed a Liver-on-a-chip (LOC) model by bioprinting the Gelatin Methacryloyl hydrogel loaded with hepatocyte extracellular vesicles (GelMA-EV) bioink onto the microfluidic chip to assess the metastasis behavior of the reprogrammed TNBC cells under the 3D liver microenvironment in vitro. Results: The combination of the genes HNF4A, FOXA2, FOXA3, ATF5, PROX1 and HNF1A could reprogram MDA-MB-231 tumor cells into human-induced hepatocytes (hiHeps), limiting metastasis of these cells. Single-cell sequencing analysis showed that the oncogenes were significantly inhibited while the liver-specific genes were activated after lineage reprogramming. Finally, the constructed LOC model showed that the hepatic phenotypes of the reprogrammed cells could be observed, and the metastasis of embedded cancer cells could be inhibited under the liver microenvironment. Conclusion: Our findings demonstrate that reprogramming could be a promising method to produce hepatocytes and treat TNBC liver metastasis. And the LOC model could intimate the 3D liver microenvironment and assess the behavior of the reprogrammed TNBC cells.
Assuntos
Neoplasias Hepáticas , Neoplasias de Mama Triplo Negativas , Humanos , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/metabolismo , Hepatócitos/metabolismo , Fatores de Transcrição/metabolismo , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Impressão Tridimensional , Dispositivos Lab-On-A-Chip , Microambiente TumoralRESUMO
Lentiviral vectors (LVs) are widely used for delivering foreign genes for long-term expression. Recently, virus-like particles (VLPs) were developed for mRNA or ribonucleoprotein (RNP) delivery for short-term endonuclease expression. Generating large amount of LVs or VLPs is challenging. On the other hand, methods for using VLPs to co-deliver Cas9 mRNA and single guide RNA (sgRNA) are limited. Fusing aptamer-binding protein (ABP) to the N-terminus of HIV Gag protein is currently the successful way to develop hybrid particles for co-delivering Cas9 mRNA and sgRNA. The effects of modifying Gag protein this way on particle assembly are unknown. Previously we found that adding an ABP after the second zinc finger domain of nucleocapsid (NC) protein had minimal effects on particle assembly. Based on these observations, here we developed hybrid particles for Cas9 mRNA and sgRNA co-delivery with normal capsid assembly efficiency. We further improved LVs for integrated gene expression by including an aptamer sequence in lentiviral genomic RNA, which improved lentiviral particle production and enhanced LV genomic RNA packaging. In summary, here we describe the development of new all-in-one VLPs for co-delivery of Cas9 mRNA and sgRNA, and new LVs for enhanced vector production and gene expression.
Assuntos
Sistemas CRISPR-Cas , RNA Guia de Cinetoplastídeos , Sistemas CRISPR-Cas/genética , Edição de Genes/métodos , Expressão Gênica , Produtos do Gene gag/genética , Oligonucleotídeos , RNA Guia de Cinetoplastídeos/genética , RNA Mensageiro/genéticaRESUMO
Type 1 Dent disease is caused by changes in chloride voltage-gated channel 5 (CLCN5) gene on chromosome X, which causes the lack or dysfunction of chloride channel ClC-5. Affected subjects show proteinuria and hypercalciuria, and eventually develop end-stage kidney disease. Currently there is no cure for this disease. Here, we used CRISPR-Cas9 technology to develop a Clcn5 mouse model with 95% of the ClC-5 coding region deleted. These mutant mice showed obvious Dent disease-like phenotypes. We used lentiviral vectors to deliver human CLCN5 cDNA into the kidneys of mutant mice by retrograde ureter injection and observed increased megalin expression, improved diuresis, and decreased urinary calcium and protein excretion, which persisted for 3 months. The therapeutic effects diminished 4 months after gene therapy. Our data suggest that immune responses to the transgene products most likely explain the loss of gene therapy effects. This study suggests that gene therapy could be a promising approach to treat Dent disease, but more work is needed to achieve sustained therapeutic effects.
RESUMO
Adeno-associated virus (AAV)-mediated clustered regularly interspaced short palindromic repeats (CRISPR) editing holds promise to restore missing dystrophin in Duchenne muscular dystrophy (DMD). Intramuscular coinjection of CRISPR-associated protein 9 (Cas9) and guide RNA (gRNA) vectors resulted in robust dystrophin restoration in short-term studies in the mdx mouse model of DMD. Intriguingly, this strategy failed to yield efficient dystrophin rescue in muscle in a long-term (18-month) systemic injection study. In-depth analyses revealed a selective loss of the gRNA vector after long-term systemic, but not short-term local injection. To determine whether preferential gRNA vector depletion is due to the mode of delivery (local vs. systemic) or the duration of the study (short term vs. long term), we conducted a short-term systemic injection study. The gRNA (4e12 vg/mouse in the 1:1 group or 1.2e13 vg/mouse in the 3:1 group) and Cas9 (4e12 vg/mouse) vectors were coinjected intravenously into 4-week-old mdx mice. The ratio of the gRNA to Cas9 vector genome copy dropped from 1:1 and 3:1 at injection to 0.4:1 and 1:1 at harvest 3 months later, suggesting that the route of administration, rather than the experimental duration, determines preferential gRNA vector loss. Consistent with our long-term systemic injection study, the vector ratio did not influence Cas9 expression. However, the 3:1 group showed significantly higher dystrophin expression and genome editing, better myofiber size distribution, and a more pronounced improvement in muscle function and electrocardiography. Our data suggest that the gRNA vector dose determines the outcome of systemic AAV CRISPR therapy for DMD.
Assuntos
Distrofina , Distrofia Muscular de Duchenne , Animais , Sistemas CRISPR-Cas/genética , Dependovirus/genética , Dependovirus/metabolismo , Distrofina/genética , Distrofina/metabolismo , Edição de Genes/métodos , Terapia Genética/métodos , Camundongos , Camundongos Endogâmicos mdx , Músculo Esquelético/metabolismo , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/terapia , RNA Guia de Cinetoplastídeos/genética , RNA Guia de Cinetoplastídeos/metabolismoRESUMO
Mitochondrial dysfunction plays an important role in mediating ischemic brain damage. Immp2l is an inner mitochondrial membrane peptidase that processes mitochondrial protein cytochrome c1 (Cyc1). Homozygous mutation of Immp2l (Immp2l(Tg(Tyr)979Ove) or Immp2l(-/-)) elevates mitochondrial membrane potential, increases superoxide (O(2)(-)) production in the brain and impairs fertility. The objectives of this study are to explore the effects of heterozygous mutation of Immp2l (Immp2l(+/-)) on ischemic outcome and to determine the influence of Immp2l deficiency on brain mitochondria after stroke. Male Immp2l(+/-) and wild-type (WT) mice were subjected to 1-h focal cerebral ischemia. Their brains were harvested after 5 and 24-h of reperfusion. The results showed that infarct volume and DNA oxidative damage significantly increased in the Immp2l(+/-) mice. There were no obvious cerebral vasculature abnormalities between the two types of mice viewed by Indian ink perfusion. The increased damage in Immp2l(+/-) mice was associated with early increase in O(2)(-) production. Mitochondrial respiratory rate, total mitochondrial respiratory capacity and mitochondrial respiratory complex activities were decreased at 5-h of recirculation in Immp2l(+/-) mice compared to WT mice. Our results suggest that Immp2l deficiency increases ischemic brain damage by enhancing O(2)(-) production and damaging mitochondrial functional performance.
Assuntos
Lesões Encefálicas/patologia , Lesões Encefálicas/fisiopatologia , Encéfalo/ultraestrutura , Endopeptidases/deficiência , Mitocôndrias/patologia , Proteínas Mitocondriais/deficiência , Análise de Variância , Animais , Encéfalo/patologia , Infarto Encefálico/etiologia , Infarto Encefálico/patologia , Lesões Encefálicas/etiologia , Isquemia Encefálica/complicações , Círculo Arterial do Cérebro/patologia , Dano ao DNA/genética , Dano ao DNA/fisiologia , Dicarbetoxi-Di-Hidrocolidina/análogos & derivados , Dicarbetoxi-Di-Hidrocolidina/metabolismo , Modelos Animais de Doenças , Masculino , Potencial da Membrana Mitocondrial/genética , Camundongos , Camundongos Knockout , Mitocôndrias/fisiologia , Consumo de Oxigênio/efeitos dos fármacos , Consumo de Oxigênio/genética , Fatores de TempoRESUMO
Transient delivery of CRISPR-based genome editing effectors is important to reduce off-target effects and immune responses. Recently extracellular vesicles (EVs) have been explored for Cas9 ribonucleoprotein (RNP) delivery. However, lack of mechanisms to enrich RNPs into EVs limited the efficiency of EVs as a RNP delivery vehicle. Here we describe a mechanism to actively enrich RNPs into EVs. We used the specific interaction between RNA aptamer and aptamer-binding protein (ABP) to enrich RNPs into EVs. We inserted RNA aptamer com into single guide RNA (sgRNA), and fused com-binding ABP Com to both termini of tetraspan protein CD63 that is abundant in exosomes. We found that the Com/com interaction enriched Cas9 and adenine base editor (ABE) RNPs into EVs, via forming a three-component complex including CD63-Com fusion protein, com-modified sgRNA and Cas9 or ABE. The RNP enriched EVs are efficient in genome editing and transiently expressed. The system is capable of delivering RNPs targeting multiple loci for multiplex genome editing. In addition, Cas9 from different species can be used together. The EV-delivered RNPs are active in vivo. The data show that the aptamer and ABP interactions can be utilized to actively enrich RNPs into EVs for improved genome editing efficiency and safety.
Assuntos
Engenharia Celular , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Vesículas Extracelulares , Edição de Genes/métodos , Veículos Farmacêuticos , Ribonucleoproteínas , Animais , Feminino , Células HEK293 , Humanos , Camundongos , Tetraspanina 30/genéticaRESUMO
Transient expression of the CRISPR-Cas9 machinery is desirable to reduce the risks of off-targets and immune responses. Electroporation of Cas9 ribonucleoproteins (RNPs) is the most common delivery method to achieve transient Cas9 expression. Recently, retroviral capsids have been used for delivering Streptococcus pyogenes Cas9 RNPs, in which Cas9 was fused to the viral proteins. The fusion strategy may cause relative low capsid assembly efficiency. We recently developed virus-like particles (VLPs) consisting of lentiviral capsid and Staphylococcus aureus Cas9 RNPs using the specific interactions between aptamer and aptamer-binding protein (ABP), and obtained near-normal capsid assembly efficiency. Here we test whether highly active Streptococcus pyogenes Cas9 (SpCas9) RNP VLPs can be generated with high efficiency by aptamer/ABP interaction. We found that by optimizing the locations and types of aptamer used for single guide RNA modification, highly active SpCas9 RNP VLPs can be generated efficiently. VLP-delivered SpCas9 generated lower off-target insertions and deletions than SpCas9 RNPs delivered by electroporation. VLPs containing Cas9 from different species and targeting multiple genes can be efficiently prepared in single-particle preparation. Multiple-target VLPs were more efficient than the combination of single-target VLPs for simultaneous targeting of multiple genes. Thus, in addition to better safety features, the Cas9 VLPs are especially suited for multiplex genome editing. In summary, our VLPs offer safe, efficient, and flexible multiplex genome editing.
Assuntos
Edição de Genes , Ribonucleoproteínas , Sistemas CRISPR-Cas/genética , Capsídeo/metabolismo , Ribonucleoproteínas/genética , Ribonucleoproteínas/metabolismo , Streptococcus pyogenes/genéticaRESUMO
Heterochronic parabiosis models have been utilized to demonstrate the role of blood-borne circulating factors in systemic effects of aging. In previous studies, heterochronic parabiosis has shown positive effects across multiple tissues in old mice. More recently, a study demonstrated old blood had a more profound negative effect on muscle performance and neurogenesis of young mice. In this study, we used heterochronic parabiosis to test the hypothesis that circulating factors mediate mitochondrial bioenergetic decline, a well-established biological hallmark of aging. We examined mitochondrial morphology, expression of mitochondrial complexes, and mitochondrial respiration from skeletal muscle of mice connected as heterochronic pairs, as well as young and old isochronic controls. Our results indicate that young heterochronic mice had significantly lower total mitochondrial content and on average had significantly smaller mitochondria compared to young isochronic controls. Expression of complex IV followed a similar pattern: young heterochronic mice had a trend for lower expression compared to young isochronic controls. Additionally, respirometric analyses indicate that young heterochronic mice had significantly lower complex I, complex I + II, and maximal mitochondrial respiration and a trend for lower complex II-driven respiration compared to young isochronic controls. Interestingly, we did not observe significant improvements in old heterochronic mice compared to old isochronic controls, demonstrating the profound deleterious effects of circulating factors from old mice on mitochondrial structure and function. We also found no significant differences between the young and old heterochronic mice, demonstrating that circulating factors can be a driver of age-related differences in mitochondrial structure and function.
Assuntos
Envelhecimento/patologia , Envelhecimento/fisiologia , Mitocôndrias/patologia , Mitocôndrias/fisiologia , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Animais , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Modelos Animais , ParabioseRESUMO
Adenine base editors (ABEs) can correct gene mutations without creating double-strand breaks. However, in recent reports, these editors showed guide-independent RNA off-target activities. This work describes our development of a delivery method to minimize ABEs' RNA off-target activity. After discovering a RNA off-target hot spot for sensitive detection of RNA off-target activities, we found that delivering ribonucleoproteins (RNPs) by electroporation generated undetectable non-specific RNA editing, but on-target base editing activity was also relatively low. We then explored a lentivirus capsid-based delivery strategy to deliver ABE. We used aptamer/aptamer-binding protein (ABP) interactions to package ABE RNPs into lentiviral capsids. Capsid RNPs were delivered to human cells for highly efficient guided base editing. Importantly, RNA off-target activities from the capsid RNPs were undetectable. Our new lentiviral capsid-based ABE RNP delivery method with minimal RNA off-target activities makes ABE one step closer to possible therapeutic applications.
Assuntos
Adenina/metabolismo , Edição de Genes/métodos , Lentivirus/genética , Edição de RNA , Ribonucleoproteínas/genética , Aptâmeros de Nucleotídeos , Sequência de Bases , Capsídeo/metabolismo , Células HEK293 , Humanos , Mutação , RNA , RNA Guia de Cinetoplastídeos/genéticaRESUMO
Generating microliver tissues to recapitulate hepatic function is of increasing importance in tissue engineering and drug screening. But the limited availability of primary hepatocytes and the marked loss of phenotype hinders their application. Human induced hepatocytes (hiHeps) generated by direct reprogramming can address the shortage of primary hepatocytes to make personalized drug prediction possible. Here, we simplify preparation of reprogramming reagents by expressing six transcriptional factors (HNF4A, FOXA2, FOXA3, ATF5, PROX1, and HNF1) from two lentiviral vectors, each expressing three factors. Transducing human fetal and adult fibroblasts with low vector dosage generated human induced hepatocyte-like cells (hiHeps) displaying characteristics of mature hepatocytes and capable of drug metabolism. To mimic the physiologic liver microenvironment and improve hepatocyte function, we prepared 3D scaffold-free microliver spheroids using hiHeps and human liver nonparenchymal cells through self-assembly without exogenous scaffolds. We then introduced the microliver spheroids into a two-organ microfluidic system to examine interactions between hepatocytes and tumor cells. The hiHeps-derived spheroids metabolized the prodrug capecitabine into the active metabolite 5-fluorouracil and induced toxicity in downstream tumor spheroids. Our results demonstrate that hiHeps can be used to make microliver spheroids and combined with a microfluidic system for drug evaluation. Our work could make it possible to use patient-specific hepatocyte-like cells to predict drug efficacy and side effects in various organs from the same patient.