RESUMO
Bacterial toxin DddA-derived cytosine base editors (DdCBEs)-composed of split DddAtox (a cytosine deaminase specific to double-stranded DNA), custom-designed TALE (transcription activator-like effector) DNA-binding proteins, and a uracil glycosylase inhibitor-enable mitochondrial DNA (mtDNA) editing in human cells, which may pave the way for therapeutic correction of pathogenic mtDNA mutations in patients. The utility of DdCBEs has been limited by off-target activity, which is probably caused by spontaneous assembly of the split DddAtox deaminase enzyme, independent of DNA-binding interactions. We engineered high-fidelity DddA-derived cytosine base editors (HiFi-DdCBEs) with minimal off-target activity by substituting alanine for amino acid residues at the interface between the split DddAtox halves. The resulting domains cannot form a functional deaminase without binding of their linked TALE proteins at adjacent sites on DNA. Whole mitochondrial genome sequencing shows that, unlike conventional DdCBEs, which induce hundreds of unwanted off-target C-to-T conversions in human mtDNA, HiFi-DdCBEs are highly efficient and precise, avoiding collateral off-target mutations, and as such, they will probably be desirable for therapeutic applications.
Assuntos
DNA Mitocondrial , Edição de Genes , Humanos , DNA Mitocondrial/genética , Edição de Genes/métodos , Mitocôndrias/metabolismo , Mutação , Citosina/metabolismo , Sistemas CRISPR-CasRESUMO
We present two methods for enhancing the efficiency of mitochondrial DNA (mtDNA) editing in mice with DddA-derived cytosine base editors (DdCBEs). First, we fused DdCBEs to a nuclear export signal (DdCBE-NES) to avoid off-target C-to-T conversions in the nuclear genome and improve editing efficiency in mtDNA. Second, mtDNA-targeted TALENs (mitoTALENs) are co-injected into mouse embryos to cleave unedited mtDNA. We generated a mouse model with the m.G12918A mutation in the MT-ND5 gene, associated with mitochondrial genetic disorders in humans. The mutant mice show hunched appearances, damaged mitochondria in kidney and brown adipose tissues, and hippocampal atrophy, resulting in premature death.
Assuntos
DNA Mitocondrial , Doenças Mitocondriais , Animais , Citosina , DNA Mitocondrial/genética , Endonucleases/metabolismo , Edição de Genes/métodos , Humanos , Camundongos , Mitocôndrias/genética , Mitocôndrias/metabolismo , Doenças Mitocondriais/genética , Sinais de Exportação Nuclear/genética , Nucleases dos Efetores Semelhantes a Ativadores de Transcrição/genéticaRESUMO
Plant organelles including mitochondria and chloroplasts contain their own genomes, which encode many genes essential for respiration and photosynthesis, respectively. Gene editing in plant organelles, an unmet need for plant genetics and biotechnology, has been hampered by the lack of appropriate tools for targeting DNA in these organelles. In this study, we developed a Golden Gate cloning system1, composed of 16 expression plasmids (8 for the delivery of the resulting protein to mitochondria and the other 8 for delivery to chloroplasts) and 424 transcription activator-like effector subarray plasmids, to assemble DddA-derived cytosine base editor (DdCBE)2 plasmids and used the resulting DdCBEs to efficiently promote point mutagenesis in mitochondria and chloroplasts. Our DdCBEs induced base editing in lettuce or rapeseed calli at frequencies of up to 25% (mitochondria) and 38% (chloroplasts). We also showed DNA-free base editing in chloroplasts by delivering DdCBE mRNA to lettuce protoplasts to avoid off-target mutations caused by DdCBE-encoding plasmids. Furthermore, we generated lettuce calli and plantlets with edit frequencies of up to 99%, which were resistant to streptomycin or spectinomycin, by introducing a point mutation in the chloroplast 16S rRNA gene.
Assuntos
Brassica napus/genética , Clonagem de Organismos/métodos , DNA de Cloroplastos , DNA Mitocondrial , Edição de Genes/métodos , Lactuca/genética , Melhoramento Vegetal/métodos , Produtos Agrícolas/genéticaRESUMO
DddA-derived cytosine base editors (DdCBEs), composed of the split interbacterial toxin DddAtox, transcription activator-like effector (TALE), and uracil glycosylase inhibitor (UGI), enable targeted C-to-T base conversions in mitochondrial DNA (mtDNA). Here, we demonstrate highly efficient mtDNA editing in mouse embryos using custom-designed DdCBEs. We target the mitochondrial gene, MT-ND5 (ND5), which encodes a subunit of NADH dehydrogenase that catalyzes NADH dehydration and electron transfer to ubiquinone, to obtain several mtDNA mutations, including m.G12918A associated with human mitochondrial diseases and m.C12336T that incorporates a premature stop codon, creating mitochondrial disease models in mice and demonstrating a potential for the treatment of mitochondrial disorders.
Assuntos
DNA Mitocondrial/genética , Edição de Genes/métodos , Genes Mitocondriais/genética , Animais , Transporte de Elétrons , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias , Doenças Mitocondriais/genética , Proteínas Mitocondriais/genética , Mutação , NADH Desidrogenase/genética , Células NIH 3T3 , Efetores Semelhantes a Ativadores de Transcrição/genéticaRESUMO
Adenine base editors (ABEs) composed of an engineered adenine deaminase and the Streptococcus pyogenes Cas9 nickase enable adenine-to-guanine (A-to-G) single-nucleotide substitutions in a guide RNA (gRNA)-dependent manner. Here we demonstrate application of this technology in mouse embryos and adult mice. We also show that long gRNAs enable adenine editing at positions one or two bases upstream of the window that is accessible with standard single guide RNAs (sgRNAs). We introduced the Himalayan point mutation in the Tyr gene by microinjecting ABE mRNA and an extended gRNA into mouse embryos, obtaining Tyr mutant mice with an albino phenotype. Furthermore, we delivered the split ABE gene, using trans-splicing adeno-associated viral vectors, to muscle cells in a mouse model of Duchenne muscular dystrophy to correct a nonsense mutation in the Dmd gene, demonstrating the therapeutic potential of base editing in adult animals.
Assuntos
Edição de Genes/métodos , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/terapia , Adenina/química , Albinismo/embriologia , Albinismo/genética , Albinismo/terapia , Animais , Sequência de Bases , Biotecnologia , DNA/genética , Modelos Animais de Doenças , Distrofina/deficiência , Distrofina/genética , Terapia Genética/métodos , Células HEK293 , Humanos , Camundongos , Camundongos Knockout , Camundongos Mutantes , Monofenol Mono-Oxigenase/genética , Reparo Gênico Alvo-Dirigido/métodosRESUMO
Base editors (BEs) composed of a cytidine deaminase fused to CRISPR-Cas9 convert cytidine to uridine, leading to single-base-pair substitutions in eukaryotic cells. We delivered BE mRNA or ribonucleoproteins targeting the Dmd or Tyr gene via electroporation or microinjection into mouse zygotes. F0 mice showed nonsense mutations with an efficiency of 44-57% and allelic frequencies of up to 100%, demonstrating an efficient method to generate mice with targeted point mutations.
Assuntos
Proteínas Associadas a CRISPR/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Embrião de Mamíferos/fisiologia , Edição de Genes/métodos , Mutagênese Sítio-Dirigida/métodos , RNA/genética , Animais , Pareamento de Bases/genética , Citidina Desaminase/genética , Embrião de Mamíferos/embriologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos ICR , Mutação Puntual/genética , Proteínas Recombinantes de Fusão/genéticaAssuntos
Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Eletroporação/métodos , Edição de Genes/métodos , Marcação de Genes/métodos , Mutagênese Sítio-Dirigida/métodos , Ribonucleoproteínas/genética , Transfecção/métodos , Animais , Camundongos , Camundongos TransgênicosRESUMO
The control of radioresistance and metastatic potential of surviving cancer cells is important for improving cancer eradication by radiotheraphy. The distal-less homeobox2 (DLX2) gene encodes for a homeobox transcription factor involved in morphogenesis and its deregulation was found in human solid tumors and hematologic malignancies. Here we investigated the role of DLX2 in association with radiation-induced epithelial to mesenchymal transition (EMT) and stem cell-like properties and its regulation by Smad2/3 signaling in irradiated A549 and MDA-MB-231 human cancer cell lines. In irradiated A549 and MDA-MB-231 cells, EMT was induced as demonstrated by EMT marker expression, phosphorylation of Smad2/3, and migratory and invasive ability. Also, irradiated A549 and MDA-MB-231 cells showed increased cancer stem cells (CSCs) marker. Interestingly, DLX2 was overexpressed upon irradiation. Therefore, we examined the role of DLX2 in radiation-induced EMT and radioresistance. The overexpression of DLX2 alone induced EMT, migration and invasion, and CSC marker expression. The reduced colony-forming ability in irradiated cells was partially restored by DLX2 overexpression. On the other hand, the depletion of DLX2 using si-RNA abolished radiation-induced EMT, CSC marker expression, and phosphorylation of Smad2/3 in irradiated A549 and MDA-MB-231 cells. Also, depletion of DLX2 increased the radiation sensitivity in both cell lines. Moreover, knockdown of Smad2/3, a key activator of TGF-ß1 pathway, abrogated the radiation-induced DLX2 expression, indicating that radiation-induced DLX2 expression is dependent on Smad2/3 signaling. These results demonstrated that DLX2 plays a crucial role in radioresistance, radiation-induced EMT and CSC marker expression, and the expression of DLX2 is regulated by Smad2/3 signaling in A549 and MDA-MB-231 cell lines.