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1.
Nat Methods ; 21(6): 983-993, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38724692

RESUMO

The inability to scalably and precisely measure the activity of developmental cis-regulatory elements (CREs) in multicellular systems is a bottleneck in genomics. Here we develop a dual RNA cassette that decouples the detection and quantification tasks inherent to multiplex single-cell reporter assays. The resulting measurement of reporter expression is accurate over multiple orders of magnitude, with a precision approaching the limit set by Poisson counting noise. Together with RNA barcode stabilization via circularization, these scalable single-cell quantitative expression reporters provide high-contrast readouts, analogous to classic in situ assays but entirely from sequencing. Screening >200 regions of accessible chromatin in a multicellular in vitro model of early mammalian development, we identify 13 (8 previously uncharacterized) autonomous and cell-type-specific developmental CREs. We further demonstrate that chimeric CRE pairs generate cognate two-cell-type activity profiles and assess gain- and loss-of-function multicellular expression phenotypes from CRE variants with perturbed transcription factor binding sites. Single-cell quantitative expression reporters can be applied in developmental and multicellular systems to quantitatively characterize native, perturbed and synthetic CREs at scale, with high sensitivity and at single-cell resolution.


Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Análise de Célula Única , Análise de Célula Única/métodos , Animais , Camundongos , Genes Reporter , Sequências Reguladoras de Ácido Nucleico , Humanos , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Cromatina/genética , Cromatina/metabolismo , Elementos Reguladores de Transcrição , Perfilação da Expressão Gênica/métodos
2.
Nature ; 608(7921): 98-107, 2022 08.
Artigo em Inglês | MEDLINE | ID: mdl-35794474

RESUMO

DNA is naturally well suited to serve as a digital medium for in vivo molecular recording. However, contemporary DNA-based memory devices are constrained in terms of the number of distinct 'symbols' that can be concurrently recorded and/or by a failure to capture the order in which events occur1. Here we describe DNA Typewriter, a general system for in vivo molecular recording that overcomes these and other limitations. For DNA Typewriter, the blank recording medium ('DNA Tape') consists of a tandem array of partial CRISPR-Cas9 target sites, with all but the first site truncated at their 5' ends and therefore inactive. Short insertional edits serve as symbols that record the identity of the prime editing guide RNA2 mediating the edit while also shifting the position of the 'type guide' by one unit along the DNA Tape, that is, sequential genome editing. In this proof of concept of DNA Typewriter, we demonstrate recording and decoding of thousands of symbols, complex event histories and short text messages; evaluate the performance of dozens of orthogonal tapes; and construct 'long tape' potentially capable of recording as many as 20 serial events. Finally, we leverage DNA Typewriter in conjunction with single-cell RNA-seq to reconstruct a monophyletic lineage of 3,257 cells and find that the Poisson-like accumulation of sequential edits to multicopy DNA tape can be maintained across at least 20 generations and 25 days of in vitro clonal expansion.


Assuntos
DNA , Edição de Genes , Genoma , Sistemas CRISPR-Cas/genética , DNA/genética , Edição de Genes/métodos , Genoma/genética , RNA Guia de Cinetoplastídeos/genética , RNA-Seq , Análise de Célula Única , Fatores de Tempo
3.
Nat Biotechnol ; 40(2): 218-226, 2022 02.
Artigo em Inglês | MEDLINE | ID: mdl-34650269

RESUMO

Current methods to delete genomic sequences are based on clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 and pairs of single-guide RNAs (sgRNAs), but can be inefficient and imprecise, with errors including small indels as well as unintended large deletions and more complex rearrangements. In the present study, we describe a prime editing-based method, PRIME-Del, which induces a deletion using a pair of prime editing sgRNAs (pegRNAs) that target opposite DNA strands, programming not only the sites that are nicked but also the outcome of the repair. PRIME-Del achieves markedly higher precision than CRISPR-Cas9 and sgRNA pairs in programming deletions up to 10 kb, with 1-30% editing efficiency. PRIME-Del can also be used to couple genomic deletions with short insertions, enabling deletions with junctions that do not fall at protospacer-adjacent motif sites. Finally, extended expression of prime editing components can substantially enhance efficiency without compromising precision. We anticipate that PRIME-Del will be broadly useful for precise, flexible programming of genomic deletions, epitope tagging and, potentially, programming genomic rearrangements.


Assuntos
Sistemas CRISPR-Cas , RNA Guia de Cinetoplastídeos , Sistemas CRISPR-Cas/genética , DNA/genética , Edição de Genes/métodos , Genoma , Genômica , RNA Guia de Cinetoplastídeos/genética , RNA Guia de Cinetoplastídeos/metabolismo
4.
Proc Natl Acad Sci U S A ; 117(10): 5394-5401, 2020 03 10.
Artigo em Inglês | MEDLINE | ID: mdl-32094176

RESUMO

As a prototype of genomics-guided precision medicine, individualized thiopurine dosing based on pharmacogenetics is a highly effective way to mitigate hematopoietic toxicity of this class of drugs. Recently, NUDT15 deficiency was identified as a genetic cause of thiopurine toxicity, and NUDT15-informed preemptive dose reduction was quickly adopted in clinical settings. To exhaustively identify pharmacogenetic variants in this gene, we developed massively parallel NUDT15 function assays to determine the variants' effect on protein abundance and thiopurine cytotoxicity. Of the 3,097 possible missense variants, we characterized the abundance of 2,922 variants and found 54 hotspot residues at which variants resulted in complete loss of protein stability. Analyzing 2,935 variants in the thiopurine cytotoxicity-based assay, we identified 17 additional residues where variants altered NUDT15 activity without affecting protein stability. We identified structural elements key to NUDT15 stability and/or catalytical activity with single amino acid resolution. Functional effects for NUDT15 variants accurately predicted toxicity risk alleles in patients treated with thiopurines with far superior sensitivity and specificity compared to bioinformatic prediction algorithms. In conclusion, our massively parallel variant function assays identified 1,152 deleterious NUDT15 variants, providing a comprehensive reference of variant function and vastly improving the ability to implement pharmacogenetics-guided thiopurine treatment individualization.


Assuntos
Antimetabólitos/administração & dosagem , Antimetabólitos/toxicidade , Mercaptopurina/administração & dosagem , Mercaptopurina/toxicidade , Variantes Farmacogenômicos , Pirofosfatases/genética , Alelos , Substituição de Aminoácidos , Relação Dose-Resposta a Droga , Determinação de Ponto Final , Estabilidade Enzimática , Células HEK293 , Humanos , Mutação de Sentido Incorreto , Medicina de Precisão , Conformação Proteica em alfa-Hélice/genética , Pirofosfatases/química , Risco
5.
Artigo em Inglês | MEDLINE | ID: mdl-29804539

RESUMO

Metabolic syndrome induces an increased cardiovascular morbidity and mortality. Most importantly, the prevalence of metabolic syndrome in adult population is expanding. Both clinical and preclinical studies indicate that increased Free Fatty Acids (FFAs) are involved in the pathogenesis of insulin resistance and subsequent development of metabolic syndrome. The relevance of FFAs in protecting and restoring tissue function is quite vast. The search to correlate the functional deterioration of the tissues within the cardiovascular system and increased plasma concentrations of FFAs has been reported. The importance of reduction in the consumption of dietary fatty acids along with the identification of dysregulated genes responsible for persistent increased FFAs uptake and mitochondrial ß-oxidation has been increasingly recognized. This review discusses the current empirical understanding of the different types of fatty acids and their metabolism and functions both in physiological and pathophysiological conditions. We also discuss in detail about the molecular and pathophysiological basis of increased FFAs, which augments Cardiovascular Disease (CVD).


Assuntos
Ácidos Graxos não Esterificados/fisiologia , Síndrome Metabólica , Humanos , Síndrome Metabólica/fisiopatologia , Ácido Oleico/sangue , Ácido Oleico/fisiologia , Ácido Palmítico/sangue , Fatores de Risco
6.
Blood ; 131(22): 2466-2474, 2018 05 31.
Artigo em Inglês | MEDLINE | ID: mdl-29572377

RESUMO

Thiopurines (eg, 6-mercaptopurine [MP]) are highly efficacious antileukemic agents, but they are also associated with dose-limiting toxicities. Recent studies by us and others have identified inherited NUDT15 deficiency as a novel genetic cause of thiopurine toxicity, and there is a strong rationale for NUDT15-guided dose individualization to preemptively mitigate adverse effects of these drugs. Using CRISPR-Cas9 genome editing, we established a Nudt15-/- mouse model to evaluate the effectiveness of this strategy in vivo. Across MP dosages, Nudt15-/- mice experienced severe leukopenia, rapid weight loss, earlier death resulting from toxicity, and more bone marrow hypocellularity compared with wild-type mice. Nudt15-/- mice also showed excessive accumulation of a thiopurine active metabolite (ie, DNA-incorporated thioguanine nucleotides [DNA-TG]) in an MP dose-dependent fashion, as a plausible cause of increased toxicity. MP dose reduction effectively normalized systemic exposure to DNA-TG in Nudt15-/- mice and largely eliminated Nudt15 deficiency-mediated toxicity. In 95 children with acute lymphoblastic leukemia, MP dose adjustment also directly led to alteration in DNA-TG levels, the effects of which were proportional to the degree of NUDT15 deficiency. Using leukemia-bearing mice with concordant Nudt15 genotype in leukemia and host, we also confirmed that therapeutic efficacy was preserved in Nudt15-/- mice receiving a reduced MP dose compared with Nudt15+/+ counterparts exposed to a standard dose. In conclusion, we demonstrated that NUDT15 genotype-guided MP dose individualization can preemptively mitigate toxicity without compromising therapeutic efficacy.


Assuntos
Antimetabólitos Antineoplásicos/uso terapêutico , Leucemia/tratamento farmacológico , Mercaptopurina/uso terapêutico , Diester Fosfórico Hidrolases/genética , Animais , Antimetabólitos Antineoplásicos/administração & dosagem , Antimetabólitos Antineoplásicos/toxicidade , Sistemas CRISPR-Cas , Criança , Cálculos da Dosagem de Medicamento , Avaliação Pré-Clínica de Medicamentos , Deleção de Genes , Edição de Genes , Genótipo , Humanos , Leucemia/genética , Leucemia/patologia , Mercaptopurina/administração & dosagem , Mercaptopurina/toxicidade , Camundongos , Camundongos Knockout , Pirofosfatases/genética
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