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1.
Bull World Health Organ ; 99(9): 616-617, 2021 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-34475598

RESUMO

Gary Humphreys talks to Kazuto Kato about the ethical and societal challenges posed by biotechnologies that allow for the editing of the human genome.


Assuntos
Temas Bioéticos , Biotecnologia/ética , Ética Médica , Edição de Genes/ética , Temas Bioéticos/história , Sistemas CRISPR-Cas , Teoria Ética , Ética Médica/história , Edição de Genes/história , História do Século XXI , Características Humanas , Humanos
3.
Nucleic Acids Res ; 49(15): 8732-8742, 2021 09 07.
Artigo em Inglês | MEDLINE | ID: mdl-34365511

RESUMO

CRISPR-Cas9 generates double-stranded DNA breaks (DSBs) to activate cellular DNA repair pathways for genome editing. The repair of DSBs leads to small insertions or deletions (indels) and other complex byproducts, including large deletions and chromosomal translocations. Indels are well understood to disrupt target genes, while the other deleterious byproducts remain elusive. We developed a new in silico analysis pipeline for the previously described primer-extension-mediated sequencing assay to comprehensively characterize CRISPR-Cas9-induced DSB repair outcomes in human or mouse cells. We identified tremendous deleterious DSB repair byproducts of CRISPR-Cas9 editing, including large deletions, vector integrations, and chromosomal translocations. We further elucidated the important roles of microhomology, chromosomal interaction, recurrent DSBs, and DSB repair pathways in the generation of these byproducts. Our findings provide an extra dimension for genome editing safety besides off-targets. And caution should be exercised to avoid not only off-target damages but also deleterious DSB repair byproducts during genome editing.


Assuntos
Proteína 9 Associada à CRISPR , Sistemas CRISPR-Cas , Reparo do DNA , Edição de Genes , Animais , Células Cultivadas , Simulação por Computador , Humanos , Camundongos , Plasmídeos/genética , Deleção de Sequência , Translocação Genética
4.
Nat Commun ; 12(1): 5090, 2021 08 24.
Artigo em Inglês | MEDLINE | ID: mdl-34429421

RESUMO

CRISPR-Cas9 mediated genome editing offers unprecedented opportunities for treating human diseases. There are several reports that demonstrate pre-existing immune responses to Cas9 which may have implications for clinical development of CRISPR-Cas9 mediated gene therapy. Here we use 209 overlapping peptides that span the entire sequence of Staphylococcus aureus Cas9 (SaCas9) and human peripheral blood mononuclear cells (PBMCs) from a cohort of donors with a distribution of Major Histocompatibility Complex (MHC) alleles comparable to that in the North American (NA) population to identify the immunodominant regions of the SaCas9 protein. We also use an MHC Associated Peptide Proteomics (MAPPs) assay to identify SaCas9 peptides presented by MHC Class II (MHC-II) proteins on dendritic cells. Using these two data sets we identify 22 SaCas9 peptides that are both presented by MHC-II proteins and stimulate CD4+ T-cells.


Assuntos
Linfócitos T CD4-Positivos/imunologia , Sistemas CRISPR-Cas , Proliferação de Células/genética , Antígenos de Histocompatibilidade Classe II/metabolismo , Peptídeos/genética , Peptídeos/metabolismo , Sequência de Aminoácidos , Proteína 9 Associada à CRISPR/genética , Citocinas , Edição de Genes , Humanos , Staphylococcus aureus/genética , Linfócitos T/imunologia
5.
Int J Mol Sci ; 22(15)2021 Jul 28.
Artigo em Inglês | MEDLINE | ID: mdl-34360834

RESUMO

Achromatopsia is characterized by amblyopia, photophobia, nystagmus, and color blindness. Previous animal models of achromatopsia have shown promising results using gene augmentation to restore cone function. However, the optimal therapeutic window to elicit recovery remains unknown. Here, we attempted two rounds of gene augmentation to generate recoverable mouse models of achromatopsia including a Cnga3 model with a knock-in stop cassette in intron 5 using Easi-CRISPR (Efficient additions with ssDNA inserts-CRISPR) and targeted embryonic stem (ES) cells. This model demonstrated that only 20% of CNGA3 levels in homozygotes derived from target ES cells remained, as compared to normal CNGA3 levels. Despite the low percentage of remaining protein, the knock-in mouse model continued to generate normal cone phototransduction. Our results showed that a small amount of normal CNGA3 protein is sufficient to form "functional" CNG channels and achieve physiological demand for proper cone phototransduction. Thus, it can be concluded that mutating the Cnga3 locus to disrupt the functional tetrameric CNG channels may ultimately require more potent STOP cassettes to generate a reversible achromatopsia mouse model. Our data also possess implications for future CNGA3-associated achromatopsia clinical trials, whereby restoration of only 20% functional CNGA3 protein may be sufficient to form functional CNG channels and thus rescue cone response.


Assuntos
Defeitos da Visão Cromática/genética , Canais de Cátion Regulados por Nucleotídeos Cíclicos/genética , Modelos Animais de Doenças , Edição de Genes , Mutação , Animais , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Defeitos da Visão Cromática/metabolismo , Técnicas de Introdução de Genes , Camundongos , Células Fotorreceptoras Retinianas Cones/metabolismo , Células Fotorreceptoras Retinianas Cones/fisiologia
6.
Int J Mol Sci ; 22(16)2021 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-34445221

RESUMO

Schizophrenia (SZ) is a prevalent functional psychosis characterized by clinical behavioural symptoms and underlying abnormalities in brain function. Genome-wide association studies (GWAS) of schizophrenia have revealed many loci that do not directly identify processes disturbed in the disease. For this reason, the development of cellular models containing SZ-associated variations has become a focus in the post-GWAS research era. The application of revolutionary clustered regularly interspaced palindromic repeats CRISPR/Cas9 gene-editing tools, along with recently developed technologies for cultivating brain organoids in vitro, have opened new perspectives for the construction of these models. In general, cellular models are intended to unravel particular biological phenomena. They can provide the missing link between schizophrenia-related phenotypic features (such as transcriptional dysregulation, oxidative stress and synaptic dysregulation) and data from pathomorphological, electrophysiological and behavioural studies. The objectives of this review are the systematization and classification of cellular models of schizophrenia, based on their complexity and validity for understanding schizophrenia-related phenotypes.


Assuntos
Sistemas CRISPR-Cas , Edição de Genes , Regulação da Expressão Gênica , Modelos Neurológicos , Esquizofrenia , Pesquisa Biomédica , Estudo de Associação Genômica Ampla , Humanos , Esquizofrenia/genética , Esquizofrenia/metabolismo
7.
Int J Mol Sci ; 22(15)2021 Jul 29.
Artigo em Inglês | MEDLINE | ID: mdl-34360895

RESUMO

BACKGROUND: Type 2 diabetes mellitus is one of the leading causes of morbidity and mortality worldwide and is derived from an accumulation of genetic and epigenetic changes. In this study, we aimed to construct Insilco, a competing endogenous RNA (ceRNA) network linked to the pathogenesis of insulin resistance followed by its experimental validation in patients', matched control and cell line samples, as well as to evaluate the efficacy of CRISPR/Cas9 as a potential therapeutic strategy to modulate the expression of this deregulated network. By applying bioinformatics tools through a two-step process, we identified and verified a ceRNA network panel of mRNAs, miRNAs and lncRNA related to insulin resistance, Then validated the expression in clinical samples (123 patients and 106 controls) and some of matched cell line samples using real time PCR. Next, two guide RNAs were designed to target the sequence flanking LncRNA/miRNAs interaction by CRISPER/Cas9 in cell culture. Gene editing tool efficacy was assessed by measuring the network downstream proteins GLUT4 and mTOR via immunofluorescence. RESULTS: LncRNA-RP11-773H22.4, together with RET, IGF1R and mTOR mRNAs, showed significant upregulation in T2DM compared with matched controls, while miRNA (i.e., miR-3163 and miR-1) and mRNA (i.e., GLUT4 and AKT2) expression displayed marked downregulation in diabetic samples. CRISPR/Cas9 successfully knocked out LncRNA-RP11-773H22.4, as evidenced by the reversal of the gene expression of the identified network at RNA and protein levels to the normal expression pattern after gene editing. CONCLUSIONS: The present study provides the significance of this ceRNA based network and its related target genes panel both in the pathogenesis of insulin resistance and as a therapeutic target for gene editing in T2DM.


Assuntos
Sistemas CRISPR-Cas , Biologia Computacional/métodos , Diabetes Mellitus Tipo 2/genética , Edição de Genes/métodos , Expressão Gênica , Resistência à Insulina/genética , MicroRNAs/genética , RNA Longo não Codificante/genética , RNA Mensageiro/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Estudos de Casos e Controles , Linhagem Celular , Diabetes Mellitus Tipo 2/sangue , Feminino , Redes Reguladoras de Genes , Hospitais Universitários , Humanos , Linfócitos/metabolismo , Masculino , Pessoa de Meia-Idade
8.
Front Immunol ; 12: 655122, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34408743

RESUMO

FOXP3+ regulatory T cells (Tregs) are central for maintaining peripheral tolerance and immune homeostasis. Because of their immunosuppressive characteristics, Tregs are a potential therapeutic target in various diseases such as autoimmunity, transplantation and infectious diseases like COVID-19. Numerous studies are currently exploring the potential of adoptive Treg therapy in different disease settings and novel genome editing techniques like CRISPR/Cas will likely widen possibilities to strengthen its efficacy. However, robust and expeditious protocols for genome editing of human Tregs are limited. Here, we describe a rapid and effective protocol for reaching high genome editing efficiencies in human Tregs without compromising cell integrity, suitable for potential therapeutic applications. By deletion of IL2RA encoding for IL-2 receptor α-chain (CD25) in Tregs, we demonstrated the applicability of the method for downstream functional assays and highlighted the importance for CD25 for in vitro suppressive function of human Tregs. Moreover, deletion of IL6RA (CD126) in human Tregs elicits cytokine unresponsiveness and thus may prevent IL-6-mediated instability of Tregs, making it an attractive target to potentially boost functionality in settings of adoptive Treg therapies to contain overreaching inflammation or autoimmunity. Thus, our rapid and efficient protocol for genome editing in human Tregs may advance possibilities for Treg-based cellular therapies.


Assuntos
Edição de Genes/métodos , Subunidade alfa de Receptor de Interleucina-2/genética , Receptores de Interleucina-6/genética , Linfócitos T Reguladores/metabolismo , Buffy Coat/citologia , Sistemas CRISPR-Cas/genética , Fatores de Transcrição Forkhead/metabolismo , Técnicas de Silenciamento de Genes , Células HEK293 , Voluntários Saudáveis , Humanos , Imunoterapia Adotiva/métodos , Cultura Primária de Células , RNA Guia/genética , Fatores de Tempo
9.
Nat Commun ; 12(1): 4902, 2021 08 12.
Artigo em Inglês | MEDLINE | ID: mdl-34385461

RESUMO

Efficient and precise base editors (BEs) for C-to-G transversion are highly desirable. However, the sequence context affecting editing outcome largely remains unclear. Here we report engineered C-to-G BEs of high efficiency and fidelity, with the sequence context predictable via machine-learning methods. By changing the species origin and relative position of uracil-DNA glycosylase and deaminase, together with codon optimization, we obtain optimized C-to-G BEs (OPTI-CGBEs) for efficient C-to-G transversion. The motif preference of OPTI-CGBEs for editing 100 endogenous sites is determined in HEK293T cells. Using a sgRNA library comprising 41,388 sequences, we develop a deep-learning model that accurately predicts the OPTI-CGBE editing outcome for targeted sites with specific sequence context. These OPTI-CGBEs are further shown to be capable of efficient base editing in mouse embryos for generating Tyr-edited offspring. Thus, these engineered CGBEs are useful for efficient and precise base editing, with outcome predictable based on sequence context of targeted sites.


Assuntos
Sistemas CRISPR-Cas , Citidina Desaminase/metabolismo , Edição de Genes/métodos , Aprendizado de Máquina , Uracila-DNA Glicosidase/metabolismo , Animais , Sequência de Bases , Sítios de Ligação/genética , Caenorhabditis elegans/genética , Códon/genética , Citidina Desaminase/genética , Escherichia coli/genética , Feminino , Biblioteca Gênica , Células HEK293 , Humanos , Camundongos , Reprodutibilidade dos Testes , Uracila-DNA Glicosidase/genética
10.
Nat Commun ; 12(1): 4922, 2021 08 13.
Artigo em Inglês | MEDLINE | ID: mdl-34389729

RESUMO

CRISPR-Cas9 is a promising technology for gene therapy. However, the ON-target genotoxicity of CRISPR-Cas9 nuclease due to DNA double-strand breaks has received little attention and is probably underestimated. Here we report that genome editing targeting globin genes induces megabase-scale losses of heterozygosity (LOH) from the globin CRISPR-Cas9 cut-site to the telomere (5.2 Mb). In established lines, CRISPR-Cas9 nuclease induces frequent terminal chromosome 11p truncations and rare copy-neutral LOH. In primary hematopoietic progenitor/stem cells, we detect 1.1% of clones (7/648) with acquired megabase LOH induced by CRISPR-Cas9. In-depth analysis by SNP-array reveals the presence of copy-neutral LOH. This leads to 11p15.5 partial uniparental disomy, comprising two Chr11p15.5 imprinting centers (H19/IGF2:IG-DMR/IC1 and KCNQ1OT1:TSS-DMR/IC2) and impacting H19 and IGF2 expression. While this genotoxicity is a safety concern for CRISPR clinical trials, it is also an opportunity to model copy-neutral-LOH for genetic diseases and cancers.


Assuntos
Sistemas CRISPR-Cas , Edição de Genes/métodos , Globinas/genética , Células-Tronco Hematopoéticas/metabolismo , Perda de Heterozigosidade/genética , Deleção de Sequência , Células Cultivadas , Deleção Cromossômica , Cromossomos Humanos Par 11/genética , Metilação de DNA , Expressão Gênica , Células HEK293 , Células-Tronco Hematopoéticas/citologia , Humanos , Fator de Crescimento Insulin-Like II/genética , Polimorfismo de Nucleotídeo Único , RNA Longo não Codificante/genética
11.
Nat Commun ; 12(1): 5078, 2021 08 23.
Artigo em Inglês | MEDLINE | ID: mdl-34426578

RESUMO

Genome-wide association studies (GWAS) have identified loci for kidney disease, but the causal variants, genes, and pathways remain unknown. Here we identify two kidney disease genes Dipeptidase 1 (DPEP1) and Charged Multivesicular Body Protein 1 A (CHMP1A) via the triangulation of kidney function GWAS, human kidney expression, and methylation quantitative trait loci. Using single-cell chromatin accessibility and genome editing, we fine map the region that controls the expression of both genes. Mouse genetic models demonstrate the causal roles of both genes in kidney disease. Cellular studies indicate that both Dpep1 and Chmp1a are important regulators of a single pathway, ferroptosis and lead to kidney disease development via altering cellular iron trafficking.


Assuntos
Dipeptidases/genética , Ferroptose/genética , Loci Gênicos , Predisposição Genética para Doença , Nefropatias/genética , Proteínas de Transporte Vesicular/genética , Animais , Nitrogênio da Ureia Sanguínea , Cromatina/metabolismo , Cisplatino , Metilação de DNA/genética , Dipeptidases/deficiência , Dipeptidases/metabolismo , Ácido Fólico , Edição de Genes , Regulação da Expressão Gênica , Estudo de Associação Genômica Ampla , Haploinsuficiência/genética , Humanos , Ferro/metabolismo , Rim/patologia , Nefropatias/induzido quimicamente , Camundongos , Necroptose/genética , Especificidade de Órgãos , Mapeamento Físico do Cromossomo , Piroptose/genética , Locos de Características Quantitativas , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Transporte Vesicular/deficiência , Proteínas de Transporte Vesicular/metabolismo
12.
Methods Mol Biol ; 2351: 321-334, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34382198

RESUMO

Loss-of-function experiments are essential for the functional investigation of cis-regulatory elements (CREs), such as transcriptional enhancers. This can be achieved with CRISPR-Cas9 using pairs of single guide RNAs (sgRNAs) to target the flanking regions of a CRE. Here, I describe a single-step protocol to rapidly and inexpensively generate vectors co-expressing two sgRNAs, which allows re-usage of gRNAs oligonucleotides from one experimental design to another. This protocol is applicable to cloning sgRNAs into virtually any CRISPR-Cas9 backbone that allows cloning using Golden Gate, by adapting the primer design.


Assuntos
Sistemas CRISPR-Cas , Edição de Genes , Marcação de Genes , Vetores Genéticos/genética , Sequências Reguladoras de Ácido Nucleico , Deleção de Sequência , Clonagem Molecular , Ordem dos Genes , Marcação de Genes/métodos , Humanos , Células Secretoras de Insulina/metabolismo , RNA Guia/química , RNA Guia/genética
13.
Nat Commun ; 12(1): 4991, 2021 08 17.
Artigo em Inglês | MEDLINE | ID: mdl-34404810

RESUMO

Key mechanisms of fetal hemoglobin (HbF) regulation and switching have been elucidated through studies of human genetic variation, including mutations in the HBG1/2 promoters, deletions in the ß-globin locus, and variation impacting BCL11A. While this has led to substantial insights, there has not been a unified understanding of how these distinct genetically-nominated elements, as well as other key transcription factors such as ZBTB7A, collectively interact to regulate HbF. A key limitation has been the inability to model specific genetic changes in primary isogenic human hematopoietic cells to uncover how each of these act individually and in aggregate. Here, we describe a single-cell genome editing functional assay that enables specific mutations to be recapitulated individually and in combination, providing insights into how multiple mutation-harboring functional elements collectively contribute to HbF expression. In conjunction with quantitative modeling and chromatin capture analyses, we illustrate how these genetic findings enable a comprehensive understanding of how distinct regulatory mechanisms can synergistically modulate HbF expression.


Assuntos
Edição de Genes , Hemoglobinas/genética , Hemoglobinas/metabolismo , Sistemas CRISPR-Cas , Cromatina , Cromossomos , Proteínas de Ligação a DNA/metabolismo , Hemoglobina Fetal/genética , Hemoglobina Fetal/metabolismo , Expressão Gênica , Globinas , Humanos , Mutação , Proteínas Repressoras , Fatores de Transcrição/metabolismo , Globinas beta/genética
14.
Nat Genet ; 53(8): 1177-1186, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34341563

RESUMO

Hereditary persistence of fetal hemoglobin (HPFH) ameliorates ß-hemoglobinopathies by inhibiting the developmental switch from γ-globin (HBG1/HBG2) to ß-globin (HBB) gene expression. Some forms of HPFH are associated with γ-globin promoter variants that either disrupt binding motifs for transcriptional repressors or create new motifs for transcriptional activators. How these variants sustain γ-globin gene expression postnatally remains undefined. We mapped γ-globin promoter sequences functionally in erythroid cells harboring different HPFH variants. Those that disrupt a BCL11A repressor binding element induce γ-globin expression by facilitating the recruitment of nuclear transcription factor Y (NF-Y) to a nearby proximal CCAAT box and GATA1 to an upstream motif. The proximal CCAAT element becomes dispensable for HPFH variants that generate new binding motifs for activators NF-Y or KLF1, but GATA1 recruitment remains essential. Our findings define distinct mechanisms through which transcription factors and their cis-regulatory elements activate γ-globin expression in different forms of HPFH, some of which are being recreated by therapeutic genome editing.


Assuntos
Fator de Ligação a CCAAT/genética , Hemoglobina Fetal/genética , Fator de Transcrição GATA1/genética , gama-Globinas/genética , Animais , Sítios de Ligação , Células COS , Sistemas CRISPR-Cas , Linhagem Celular , Chlorocebus aethiops , Células Eritroides , Edição de Genes/métodos , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Regiões Promotoras Genéticas , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo
15.
Science ; 373(6557): 882-889, 2021 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-34413232

RESUMO

Eukaryotic genomes contain domesticated genes from integrating viruses and mobile genetic elements. Among these are homologs of the capsid protein (known as Gag) of long terminal repeat (LTR) retrotransposons and retroviruses. We identified several mammalian Gag homologs that form virus-like particles and one LTR retrotransposon homolog, PEG10, that preferentially binds and facilitates vesicular secretion of its own messenger RNA (mRNA). We showed that the mRNA cargo of PEG10 can be reprogrammed by flanking genes of interest with Peg10's untranslated regions. Taking advantage of this reprogrammability, we developed selective endogenous encapsidation for cellular delivery (SEND) by engineering both mouse and human PEG10 to package, secrete, and deliver specific RNAs. Together, these results demonstrate that SEND is a modular platform suited for development as an efficient therapeutic delivery modality.


Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Proteínas de Ligação a DNA/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Animais , Proteínas Reguladoras de Apoptose/química , Proteínas Reguladoras de Apoptose/genética , Capsídeo/metabolismo , Linhagem Celular , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Vesículas Extracelulares/metabolismo , Edição de Genes , Vetores Genéticos , Humanos , Camundongos , Neurônios/metabolismo , Domínios Proteicos , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/genética , Retroelementos , Transfecção , Regiões não Traduzidas , Regulação para Cima
16.
Int J Mol Sci ; 22(16)2021 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-34445458

RESUMO

CRISPR/Cas, one of the most rapidly developing technologies in the world, has been applied successfully in plant science. To test new nucleases, gRNA expression systems and other inventions in this field, several plant genes with visible phenotypic effects have been constantly used as targets. Anthocyanin pigmentation is one of the most easily identified traits, that does not require any additional treatment. It is also associated with stress resistance, therefore plants with edited anthocyanin genes might be of interest for agriculture. Phenotypic effect of CRISPR/Cas editing of PAP1 and its homologs, DFR, F3H and F3'H genes have been confirmed in several distinct plant species. DFR appears to be a key structural gene of anthocyanin biosynthesis, controlled by various transcription factors. There are still many promising potential model genes that have not been edited yet. Some of them, such as Delila, MYB60, HAT1, UGT79B2, UGT79B3 and miR156, have been shown to regulate drought tolerance in addition to anthocyanin biosynthesis. Genes, also involved in trichome development, such as TTG1, GLABRA2, MYBL2 and CPC, can provide increased visibility. In this review successful events of CRISPR/Cas editing of anthocyanin genes are summarized, and new model genes are proposed. It can be useful for molecular biologists and genetic engineers, crop scientists, plant genetics and physiologists.


Assuntos
Antocianinas , Edição de Genes , Genes de Plantas , Modelos Genéticos , Plantas , Antocianinas/biossíntese , Antocianinas/genética , Plantas/genética , Plantas/metabolismo
17.
Int J Mol Sci ; 22(15)2021 Jul 28.
Artigo em Inglês | MEDLINE | ID: mdl-34360856

RESUMO

Indigenous communities across the globe, especially in rural areas, consume locally available plants known as Traditional Food Plants (TFPs) for their nutritional and health-related needs. Recent research shows that many TFPs are highly nutritious as they contain health beneficial metabolites, vitamins, mineral elements and other nutrients. Excessive reliance on the mainstream staple crops has its own disadvantages. Traditional food plants are nowadays considered important crops of the future and can act as supplementary foods for the burgeoning global population. They can also act as emergency foods in situations such as COVID-19 and in times of other pandemics. The current situation necessitates locally available alternative nutritious TFPs for sustainable food production. To increase the cultivation or improve the traits in TFPs, it is essential to understand the molecular basis of the genes that regulate some important traits such as nutritional components and resilience to biotic and abiotic stresses. The integrated use of modern omics and gene editing technologies provide great opportunities to better understand the genetic and molecular basis of superior nutrient content, climate-resilient traits and adaptation to local agroclimatic zones. Recently, realizing the importance and benefits of TFPs, scientists have shown interest in the prospection and sequencing of TFPs for their improvements, cultivation and mainstreaming. Integrated omics such as genomics, transcriptomics, proteomics, metabolomics and ionomics are successfully used in plants and have provided a comprehensive understanding of gene-protein-metabolite networks. Combined use of omics and editing tools has led to successful editing of beneficial traits in several TFPs. This suggests that there is ample scope for improvement of TFPs for sustainable food production. In this article, we highlight the importance, scope and progress towards improvement of TFPs for valuable traits by integrated use of omics and gene editing techniques.


Assuntos
Segurança Alimentar/métodos , Plantas Comestíveis/genética , Plantas Comestíveis/metabolismo , Edição de Genes , Genômica/métodos , Humanos , Metabolômica , Plantas Comestíveis/química , Proteômica
18.
BMC Plant Biol ; 21(1): 363, 2021 Aug 10.
Artigo em Inglês | MEDLINE | ID: mdl-34376154

RESUMO

BACKGROUND: To meet increasing demand for forest-based products and protect natural forests from further deforestation requires increased productivity from planted forests. Genetic improvement of conifers by traditional breeding is time consuming due to the long juvenile phase and genome complexity. Genetic modification (GM) offers the opportunity to make transformational changes in shorter time frames but is challenged by current genetically modified organism (GMO) regulations. Genome editing, which can be used to generate site-specific mutations, offers the opportunity to rapidly implement targeted improvements and is globally regulated in a less restrictive way than GM technologies. RESULTS: We have demonstrated CRISPR/Cas9 genome editing in P. radiata targeting a single-copy cell wall gene GUX1 in somatic embryogenic tissue and produced plantlets from the edited tissue. We generated biallelic INDELs with an efficiency of 15 % using a single gRNA. 12 % of the transgenic embryogenic tissue was edited when two gRNAs were used and deletions of up to 1.3 kb were identified. However, the regenerated plants did not contain large deletions but had single nucleotide insertions at one of the target sites. We assessed the use of CRISPR/Cas9 ribonucleoproteins (RNPs) for their ability to accomplish DNA-free genome editing in P. radiata. We chose a hybrid approach, with RNPs co-delivered with a plasmid-based selectable marker. A two-gRNA strategy was used which produced an editing efficiency of 33 %, and generated INDELs, including large deletions. Using the RNP approach, deletions found in embryogenic tissue were also present in the plantlets. But, all plants produced using the RNP strategy were monoallelic. CONCLUSIONS: We have demonstrated the generation of biallelic and monoallelic INDELs in the coniferous tree P. radiata with the CRISPR/Cas9 system using plasmid expressed Cas9 gRNA and RNPs respectively. This opens the opportunity to apply genome editing in conifers to rapidly modify key traits of interest.


Assuntos
Sistemas CRISPR-Cas , Edição de Genes/métodos , Genoma de Planta , Pinus/genética , DNA de Plantas , Mutação INDEL , RNA Guia , Ribonucleoproteínas/genética
19.
Nat Commun ; 12(1): 4744, 2021 08 06.
Artigo em Inglês | MEDLINE | ID: mdl-34362895

RESUMO

Human induced pluripotent stem cells (iPSC) hold promise for modeling diseases in individual human genetic backgrounds and thus for developing precision medicine. Here, we generate sensorimotor organoids containing physiologically functional neuromuscular junctions (NMJs) and apply the model to different subgroups of amyotrophic lateral sclerosis (ALS). Using a range of molecular, genomic, and physiological techniques, we identify and characterize motor neurons and skeletal muscle, along with sensory neurons, astrocytes, microglia, and vasculature. Organoid cultures derived from multiple human iPSC lines generated from individuals with ALS and isogenic lines edited to harbor familial ALS mutations show impairment at the level of the NMJ, as detected by both contraction and immunocytochemical measurements. The physiological resolution of the human NMJ synapse, combined with the generation of major cellular cohorts exerting autonomous and non-cell autonomous effects in motor and sensory diseases, may prove valuable to understand the pathophysiological mechanisms of ALS.


Assuntos
Esclerose Amiotrófica Lateral/metabolismo , Junção Neuromuscular/metabolismo , Organoides/fisiologia , Esclerose Amiotrófica Lateral/genética , Esclerose Amiotrófica Lateral/patologia , Astrócitos , Edição de Genes , Humanos , Células-Tronco Pluripotentes Induzidas , Neurônios Motores , Células Musculares , Músculo Esquelético , Mutação , Organoides/patologia , Células-Tronco
20.
Int J Mol Sci ; 22(15)2021 Jul 27.
Artigo em Inglês | MEDLINE | ID: mdl-34360757

RESUMO

Thyroid cancer is the most common endocrine malignancy, and the characterization of the genetic alterations in coding-genes that drive thyroid cancer are well consolidated in MAPK signaling. In the context of non-coding RNAs, microRNAs (miRNAs) are small non-coding RNAs that, when deregulated, cooperate to promote tumorigenesis by targeting mRNAs, many of which are proto-oncogenes and tumor suppressors. In thyroid cancer, miR-146b-5p is the most overexpressed miRNA associated with tumor aggressiveness and progression, while the antisense blocking of miR-146b-5p results in anti-tumoral effect. Therefore, inactivating miR-146b has been considered as a promising strategy in thyroid cancer therapy. Here, we applied the CRISPR/Cas9n editing system to target the MIR146B gene in an aggressive anaplastic thyroid cancer (ATC) cell line. For that, we designed two single-guide RNAs cloned into plasmids to direct Cas9 nickase (Cas9n) to the genomic region of the pre-mir-146b structure to target miR-146b-5p and miR-146b-3p sequences. In this plasmidial strategy, we cotransfected pSp-Cas9n-miR-146b-GuideA-puromycin and pSp-Cas9n-miR-146b-GuideB-GFP plasmids in KTC2 cells and selected the puromycin resistant + GFP positive clones (KTC2-Cl). As a result, we observed that the ATC cell line KTC2-Cl1 showed a 60% decrease in the expression of miR-146b-5p compared to the control, also showing reduced cell viability, migration, colony formation, and blockage of tumor development in immunocompromised mice. The analysis of the MIR146B edited sequence shows a 5 nt deletion in the miR-146b-5p region and a 1 nt deletion in the miR-146b-3p region in KTC2-Cl1. Thus, we developed an effective CRISPR/Cas9n system to edit the MIR146B miRNA gene and reduce miR-146b-5p expression which constitutes a potential molecular tool for the investigation of miRNAs function in thyroid cancer.


Assuntos
Sistemas CRISPR-Cas , Edição de Genes , Marcação de Genes , MicroRNAs , RNA Neoplásico , Carcinoma Anaplásico da Tireoide , Neoplasias da Glândula Tireoide , Animais , Linhagem Celular , Movimento Celular/genética , Sobrevivência Celular/genética , Xenoenxertos , Humanos , Camundongos , MicroRNAs/biossíntese , MicroRNAs/genética , Transplante de Neoplasias , RNA Neoplásico/biossíntese , RNA Neoplásico/genética , Carcinoma Anaplásico da Tireoide/genética , Carcinoma Anaplásico da Tireoide/metabolismo , Carcinoma Anaplásico da Tireoide/patologia , Neoplasias da Glândula Tireoide/genética , Neoplasias da Glândula Tireoide/metabolismo , Neoplasias da Glândula Tireoide/patologia
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