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Functional investigation of CRISPR-Engineered synonymous DNMT3A mutations in clonal dominance of hematopoiesis
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Resumo: As mutacoes sinonimas, historicamente consideradas neutras do ponto de vista biologico, tem vindo a ser cada vez mais reconhecidas como moduladores da expressao genica e potenciais contribuintes para a patogenese de varias doencas. No contexto da hematopoiese clonal de potencial indeterminado (CHIP), o gene DNMT3A, codificador de uma DNA metiltransferase essencial, e um dos mais frequentemente mutados, incluindo a nivel de posicoes sinonimas. No entanto, as consequencias regulatorias destas variantes permanecem, em grande parte, por esclarecer. Neste estudo, foi realizada uma caracterizacao funcional de mutacoes sinonimas em DNMT3A, combinando abordagens de edicao genomica via CRISPR/Cas9 em células monociticas humanas THP-1 com ensaios de sobre-expressao para analise da estabilidade do mRNA. O protocolo de nucleofeccao foi sistematicamente optimizado para celulas hematopoieticas em suspensao, tendo sido identificado o programa FF100 como o mais eficaz, conciliando eficiencia de transfecao com viabilidade celular. Apesar da tentativa de aumentar a eficiencia da reparacao dirigida por homologia (HDR) atraves de sincronizacao do ciclo celular com nocodazol, a maioria dos eventos de reparacao ocorreu por juncao de extremidades nao homologas (NHEJ). Ainda assim, foi possivel isolar um clone monoalelico com uma mutacao sinonima derivada de doente, bem como multiplas linhas knockout. A analise por Western blot e ensaios de atividade enzimatica confirmaram a perda completa de proteina e funcao em determinados clones, enquanto outros revelaram formas hipomorficas ou parcialmente funcionais de DNMT3A. Estes efeitos ao nivel proteico nao foram sempre previsiveis com base apenas nas alteracoes genomicas, sublinhando a necessidade de validacao funcional direta. A caracterizacao transcricional por RT-qPCR demonstrou que mutacoes sinonimas em DNMT3A podem modular significativamente a abundancia e estabilidade do mRNA, com variantes individuais a promoverem tanto o aumento como a reducao da estabilidade transcricional em relacao ao alelo selvagem. Uma analise de curta duracao (2 horas) apos bloqueio transcricional permitiu identificar efeitos agudos de destabilizacao, que se encontravam parcialmente mascarados em ensaios de maior duracao. Estes resultados constituem evidencia direta de que substituicoes sinonimas em DNMT3A tem efeitos funcionais a nivel pos-transcricional, desafiando a tradicional presuncao de neutralidade destas variantes. Atraves da integracao de engenharia genomica com perfis de estabilidade de mRNA de alta resolucao, este trabalho amplia o espectro funcional de mutações relevantes em DNMT3A no contexto da hematopoiese clonal e destaca a importancia de mecanismos regulatorios centrados no mRNA em doencas hematologicas.
Abstract: Synonymous mutations, once presumed to be biologically silent, are increasingly implicated in the regulation of gene expression and disease pathogenesis. In clonal hematopoiesis of indeterminate potential (CHIP), the DNA methyltransferase DNMT3A is one of the most frequently mutated genes, including at synonymous positions. However, the regulatory consequences of such variants remain largely uncharacterized. In this study, we functionally interrogated synonymous DNMT3A mutations using a dual approach: CRISPR/Cas9-mediated genome editing in THP-1 monocytic cells and overexpression-based transcript decay assays. Genome editing workflows were systematically optimized for suspension hematopoietic cells, identifying the FF100 nucleofection program as optimal for balancing transfection efficiency and cell viability. While homology-directed repair (HDR) remained inefficient despite nocodazole-mediated synchronization, a monoallelic clone bearing a patient-derived synonymous mutation was successfully isolated alongside multiple knockout lines. Western blot and enzymatic assays confirmed almost complete protein and functional loss in specific clones, while others retained partially functional or even hypomorphic DNMT3A variants. Notably, these protein-level effects were not always predictable from genomic edits alone, underscoring the need for direct protein validation. Transcriptomic analysis via RT-qPCR demonstrated that synonymous DNMT3A mutations can significantly modulate mRNA abundance and stability, with individual variants accelerating or delaying transcript decay relative to wildtype. A short 2-hour transcriptional arrest time course further resolved acute stability effects that were masked in longer assays. These findings provide direct evidence that synonymous substitutions in DNMT3A exert functional regulatory effects at the posttranscriptional level, challenging the long-standing assumption of their neutrality. By coupling genome engineering with high-resolution RNA stability profiling, this study expands the functional spectrum of DNMT3A mutations relevant to CHIP and highlights the importance of mRNA-centric regulatory mechanisms in hematopoietic disease.
Abstract: Synonymous mutations, once presumed to be biologically silent, are increasingly implicated in the regulation of gene expression and disease pathogenesis. In clonal hematopoiesis of indeterminate potential (CHIP), the DNA methyltransferase DNMT3A is one of the most frequently mutated genes, including at synonymous positions. However, the regulatory consequences of such variants remain largely uncharacterized. In this study, we functionally interrogated synonymous DNMT3A mutations using a dual approach: CRISPR/Cas9-mediated genome editing in THP-1 monocytic cells and overexpression-based transcript decay assays. Genome editing workflows were systematically optimized for suspension hematopoietic cells, identifying the FF100 nucleofection program as optimal for balancing transfection efficiency and cell viability. While homology-directed repair (HDR) remained inefficient despite nocodazole-mediated synchronization, a monoallelic clone bearing a patient-derived synonymous mutation was successfully isolated alongside multiple knockout lines. Western blot and enzymatic assays confirmed almost complete protein and functional loss in specific clones, while others retained partially functional or even hypomorphic DNMT3A variants. Notably, these protein-level effects were not always predictable from genomic edits alone, underscoring the need for direct protein validation. Transcriptomic analysis via RT-qPCR demonstrated that synonymous DNMT3A mutations can significantly modulate mRNA abundance and stability, with individual variants accelerating or delaying transcript decay relative to wildtype. A short 2-hour transcriptional arrest time course further resolved acute stability effects that were masked in longer assays. These findings provide direct evidence that synonymous substitutions in DNMT3A exert functional regulatory effects at the posttranscriptional level, challenging the long-standing assumption of their neutrality. By coupling genome engineering with high-resolution RNA stability profiling, this study expands the functional spectrum of DNMT3A mutations relevant to CHIP and highlights the importance of mRNA-centric regulatory mechanisms in hematopoietic disease.
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Mutations DNA methyltransferase DNMT3A CRISPR/Cas9