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An obligate step in the life cycle of HIV-1 and other retroviruses is the establishment of the provirus in target cell chromosomes. Transcriptional regulation of proviruses is complex, and understanding the mechanisms underlying this regulation has ramifications for fundamental biology, human health, and gene therapy implementation. The three core components of the Human Silencing Hub (HUSH) complex, TASOR, MPHOSPH8 (MPP8), and PPHLN1 (Periphilin 1), were identified in forward genetic screens for host genes that repress provirus expression. Subsequent loss-of-function screens revealed accessory proteins that collaborate with the HUSH complex to silence proviruses in particular contexts. To identify proteins associated with a HUSH complex-repressed provirus in human cells, we developed a technique, Provirus Proximal Proteomics, based on proximity labeling with C-BERST (dCas9-APEX2 biotinylation at genomic elements by restricted spatial tagging). Our screen exploited a lentiviral reporter that is silenced by the HUSH complex in a manner that is independent of the integration site in chromatin. Our data reveal that proviruses silenced by the HUSH complex are associated with DNA repair, mRNA processing, and transcriptional silencing proteins, including L3MBTL2, a member of the non-canonical polycomb repressive complex 1.6 (PRC1.6). A forward genetic screen confirmed that PRC1.6 components L3MBTL2 and MGA contribute to HUSH complex-mediated silencing. PRC1.6 was then shown to silence HUSH-sensitive proviruses in a promoter-specific manner. Genome wide profiling showed striking colocalization of the PRC1.6 and HUSH complexes on chromatin, primarily at sites of active promoters. Finally, PRC1.6 binding at a subset of genes that are silenced by the HUSH complex was dependent on the core HUSH complex component MPP8. These studies offer new tools with great potential for studying the transcriptional regulation of proviruses and reveal crosstalk between the HUSH complex and PRC1.6.
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Ex vivo resting culture is a standard procedure following genome editing in hematopoietic stem and progenitor cells (HSPCs). However, prolonged culture may critically affect cell viability and stem cell function. We investigated whether varying durations of culture resting times impact the engraftment efficiency of human CD34+ HSPCs edited at the BCL11A enhancer, a key regulator in the expression of fetal hemoglobin. We employed electroporation to introduce CRISPR-Cas9 components for BCL11A enhancer editing and compared outcomes with nonelectroporated (NEP) and electroporated-only (EP) control groups. Post-electroporation, we monitored cell viability, death rates, and the frequency of enriched hematopoietic stem cell (HSC) fractions (CD34+CD90+CD45RA- cells) over a 48-hour period. Our findings reveal that while the NEP group showed an increase in cell numbers 24 hours post-electroporation, both EP and BCL11A-edited groups experienced significant cell loss. Although CD34+ cell frequency remained high in all groups for up to 48 hours post-electroporation, the frequency of the HSC-enriched fraction was significantly lower in the EP and edited groups compared to the NEP group. In NBSGW xenograft mouse models, both conditioned with busulfan and nonconditioned, we found that immediate transplantation post-electroporation led to enhanced engraftment without compromising editing efficiency. Human glycophorin A+ (GPA+) red blood cells (RBCs) sorted from bone marrow of all BCL11A edited mice exhibited similar levels of γ-globin expression, regardless of infusion time. Our findings underscore the critical importance of optimizing the culture duration between genome editing and transplantation. Minimizing this interval may significantly enhance engraftment success and minimize cell loss without compromising editing efficiency. These insights offer a pathway to improve the success rates of genome editing in HSPCs, particularly for conditions like sickle cell disease.
Assuntos
Edição de Genes , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas , Animais , Humanos , Edição de Genes/métodos , Camundongos , Células-Tronco Hematopoéticas/metabolismo , Células-Tronco Hematopoéticas/citologia , Transplante de Células-Tronco Hematopoéticas/métodos , Sistemas CRISPR-Cas/genética , Eletroporação/métodos , Xenoenxertos , Sobrevivência Celular , Antígenos CD34/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismoRESUMO
OBJECTIVES: Nuclear receptor interacting protein 1 (NRIP1) suppresses energy expenditure via repression of nuclear receptors, and its depletion markedly elevates uncoupled respiration in mouse and human adipocytes. We tested whether NRIP1 deficient adipocytes implanted into obese mice would enhance whole body metabolism. Since ß-adrenergic signaling through cAMP strongly promotes adipocyte thermogenesis, we tested whether the effects of NRIP1 knock-out (NRIP1KO) require the cAMP pathway. METHODS: NRIP1KO adipocytes were implanted in recipient high-fat diet (HFD) fed mice and metabolic cage studies conducted. The Nrip1 gene was disrupted by CRISPR in primary preadipocytes isolated from control vs adipose selective GsαKO (cAdGsαKO) mice prior to differentiation to adipocytes. Protein kinase A inhibitor was also used. RESULTS: Implanting NRIP1KO adipocytes into HFD fed mice enhanced whole-body glucose tolerance by increasing insulin sensitivity, reducing adiposity, and enhancing energy expenditure in the recipients. NRIP1 depletion in both control and GsαKO adipocytes was equally effective in upregulating uncoupling protein 1 (UCP1) and adipocyte beiging, while ß-adrenergic signaling by CL 316,243 was abolished in GsαKO adipocytes. Combining NRIP1KO with CL 316,243 treatment synergistically increased Ucp1 gene expression and increased the adipocyte subpopulation responsive to beiging. Estrogen-related receptor α (ERRα) was dispensable for UCP1 upregulation by NRIPKO. CONCLUSIONS: The thermogenic effect of NRIP1 depletion in adipocytes causes systemic enhancement of energy expenditure when such adipocytes are implanted into obese mice. Furthermore, NRIP1KO acts independently but cooperatively with the cAMP pathway in mediating its effect on adipocyte beiging.
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Adipócitos , Transdução de Sinais , Camundongos , Humanos , Animais , Proteína 1 de Interação com Receptor Nuclear/metabolismo , Camundongos Obesos , Adipócitos/metabolismo , Obesidade/metabolismo , Termogênese/genéticaRESUMO
Gene editing the BCL11A erythroid enhancer is a validated approach to fetal hemoglobin (HbF) induction for ß-hemoglobinopathy therapy, though heterogeneity in edit allele distribution and HbF response may impact its safety and efficacy. Here we compared combined CRISPR-Cas9 endonuclease editing of the BCL11A +58 and +55 enhancers with leading gene modification approaches under clinical investigation. We found that combined targeting of the BCL11A +58 and +55 enhancers with 3xNLS-SpCas9 and two sgRNAs resulted in superior HbF induction, including in engrafting erythroid cells from sickle cell disease (SCD) patient xenografts, attributable to simultaneous disruption of core half E-box/GATA motifs at both enhancers. We corroborated prior observations that double strand breaks (DSBs) could produce unintended on- target outcomes in hematopoietic stem and progenitor cells (HSPCs) such as long deletions and centromere-distal chromosome fragment loss. We show these unintended outcomes are a byproduct of cellular proliferation stimulated by ex vivo culture. Editing HSPCs without cytokine culture bypassed long deletion and micronuclei formation while preserving efficient on-target editing and engraftment function. These results indicate that nuclease editing of quiescent hematopoietic stem cells (HSCs) limits DSB genotoxicity while maintaining therapeutic potency and encourages efforts for in vivo delivery of nucleases to HSCs.
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CRISPR-Cas9-based therapeutic genome editing approaches hold promise to cure a variety of human diseases. Recent findings demonstrate pre-existing immunity for the commonly used Cas orthologs from Streptococcus pyogenes (SpCas9) and Staphylococcus aureus (SaCas9) in humans, which threatens the success of this powerful tool in clinical use. Thus, a comprehensive investigation and potential risk assessment are required to exploit the full potential of the system. Here, we investigated existence of immunity to SpCas9 and SaCas9 in control rhesus macaques (Macaca mulatta) alongside monkeys transplanted with either lentiviral transduced or CRISPR-SpCas9 ribonucleoprotein (RNP)-edited cells. We observed significant levels of Cas9 antibodies in the peripheral blood of all transplanted and non-transplanted control animals. Transplantation of ex vivo transduced or SpCas9-mediated BCL11A enhancer-edited cells did not alter the levels of Cas9 antibodies in rhesus monkeys. Following stimulation of peripheral blood cells with SpCas9 or SaCas9, neither Cas9-specific T cells nor cytokine induction were detected. Robust and durable editing frequencies and expression of high levels of fetal hemoglobin in BCL11A enhancer-edited rhesus monkeys with no evidence of an immune response (>3 years) provide an optimistic outlook for the use of ex vivo CRISPR-SpCas9 (RNP)-edited cells.
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Prime editing systems have enabled the incorporation of precise edits within a genome without introducing double strand breaks. Previous studies defined an optimal primer binding site (PBS) length for the pegRNA of â¼13 nucleotides depending on the sequence composition. However, optimal PBS length characterization has been based on prime editing outcomes using plasmid or lentiviral expression systems. In this study, we demonstrate that for prime editor (PE) ribonucleoprotein complexes, the auto-inhibitory interaction between the PBS and the spacer sequence affects pegRNA binding efficiency and target recognition. Destabilizing this auto-inhibitory interaction by reducing the complementarity between the PBS-spacer region enhances prime editing efficiency in multiple prime editing formats. In the case of end-protected pegRNAs, a shorter PBS length with a PBS-target strand melting temperature near 37°C is optimal in mammalian cells. Additionally, a transient cold shock treatment of the cells post PE-pegRNA delivery further increases prime editing outcomes for pegRNAs with optimized PBS lengths. Finally, we show that prime editor ribonucleoprotein complexes programmed with pegRNAs designed using these refined parameters efficiently correct disease-related genetic mutations in patient-derived fibroblasts and efficiently install precise edits in primary human T cells and zebrafish.
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Temperatura Baixa , Edição de Genes , Peixe-Zebra , Animais , Humanos , Sítios de Ligação , Resposta ao Choque Frio , Sistemas CRISPR-Cas , Mamíferos , Ribonucleoproteínas , Peixe-Zebra/genéticaRESUMO
CRISPR gene editing holds great promise to modify DNA sequences in somatic cells to treat disease. However, standard computational and biochemical methods to predict off-target potential focus on reference genomes. We developed an efficient tool called CRISPRme that considers single-nucleotide polymorphism (SNP) and indel genetic variants to nominate and prioritize off-target sites. We tested the software with a BCL11A enhancer targeting guide RNA (gRNA) showing promise in clinical trials for sickle cell disease and ß-thalassemia and found that the top candidate off-target is produced by an allele common in African-ancestry populations (MAF 4.5%) that introduces a protospacer adjacent motif (PAM) sequence. We validated that SpCas9 generates strictly allele-specific indels and pericentric inversions in CD34+ hematopoietic stem and progenitor cells (HSPCs), although high-fidelity Cas9 mitigates this off-target. This report illustrates how genetic variants should be considered as modifiers of gene editing outcomes. We expect that variant-aware off-target assessment will become integral to therapeutic genome editing evaluation and provide a powerful approach for comprehensive off-target nomination.
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Sistemas CRISPR-Cas , Edição de Genes , Humanos , Edição de Genes/métodos , Sistemas CRISPR-Cas/genética , Células-Tronco Hematopoéticas , Mutação INDEL , RNA Guia de Sistemas CRISPR-CasRESUMO
Type V CRISPR-Cas12a systems are an attractive Cas9-alternative nuclease platform for specific genome editing applications. However, previous studies demonstrate that there is a gap in overall activity between Cas12a and Cas9 in primary cells.1 Here we describe optimization to the NLS composition and architecture of Cas12a to facilitate highly efficient targeted mutagenesis in human transformed cell lines (HEK293T, Jurkat, and K562 cells) and primary cells (NK cells and CD34+ HSPCs), regardless of Cas12a ortholog. Our 3xNLS Cas12a architecture resulted in the most robust editing platform. The improved editing activity of Cas12a in both NK cells and CD34+ HSPCs resulted in pronounced phenotypic changes associated with target gene editing. Lastly, we demonstrated that optimization of the NLS composition and architecture of Cas12a did not increase editing at potential off-target sites in HEK293T or CD34+ HSPCs. Our new Cas12a NLS variant provides an improved nuclease platform for therapeutic genome editing.
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Tay-Sachs disease (TSD) is a fatal neurodegenerative disorder caused by a deficiency of the enzyme hexosaminidase A (HexA). TSD also occurs in sheep, the only experimental model of TSD that has clinical signs of disease. The natural history of sheep TSD was characterized using serial neurological evaluations, 7 Tesla magnetic resonance imaging, echocardiograms, electrodiagnostics, and cerebrospinal fluid biomarkers. Intracranial gene therapy was also tested using AAVrh8 monocistronic vectors encoding the α-subunit of Hex (TSD α) or a mixture of two vectors encoding both the α and ß subunits separately (TSD α + ß) injected at high (1.3 × 1013 vector genomes) or low (4.2 × 1012 vector genomes) dose. Delay of symptom onset and/or reduction of acquired symptoms were noted in all adeno-associated virus-treated sheep. Postmortem evaluation showed superior HexA and vector genome distribution in the brain of TSD α + ß sheep compared to TSD α sheep, but spinal cord distribution was low in all groups. Isozyme analysis showed superior HexA formation after treatment with both vectors (TSD α + ß), and ganglioside clearance was most widespread in the TSD α + ß high-dose sheep. Microglial activation and proliferation in TSD sheep-most prominent in the cerebrum-were attenuated after gene therapy. This report demonstrates therapeutic efficacy for TSD in the sheep brain, which is on the same order of magnitude as a child's brain.
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Dependovirus , Terapia Genética , Doença de Tay-Sachs/terapia , Cadeia alfa da beta-Hexosaminidase/biossíntese , Cadeia beta da beta-Hexosaminidase/biossíntese , Animais , Encéfalo/diagnóstico por imagem , Encéfalo/enzimologia , Modelos Animais de Doenças , Ecocardiografia , Humanos , Imageamento por Ressonância Magnética , Microglia/enzimologia , Ovinos , Doença de Tay-Sachs/diagnóstico por imagem , Doença de Tay-Sachs/enzimologia , Doença de Tay-Sachs/genética , Cadeia alfa da beta-Hexosaminidase/genética , Cadeia beta da beta-Hexosaminidase/genéticaRESUMO
Adeno-associated viral (AAV) vectors have shown promise as a platform for gene therapy of neurological disorders. Achieving global gene delivery to the central nervous system (CNS) is key for development of effective therapies for many of these diseases. Here we report the isolation of a novel CNS tropic AAV capsid, AAV-B1, after a single round of in vivo selection from an AAV capsid library. Systemic injection of AAV-B1 vector in adult mice and cat resulted in widespread gene transfer throughout the CNS with transduction of multiple neuronal subpopulations. In addition, AAV-B1 transduces muscle, ß-cells, pulmonary alveoli, and retinal vasculature at high efficiency. This vector is more efficient than AAV9 for gene delivery to mouse brain, spinal cord, muscle, pancreas, and lung. Together with reduced sensitivity to neutralization by antibodies in pooled human sera, the broad transduction profile of AAV-B1 represents an important improvement over AAV9 for CNS gene therapy.