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1.
Int J Mol Sci ; 22(12)2021 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-34204013

RESUMO

Plant immunity is mediated in large part by specific interactions between a host resistance protein and a pathogen effector protein, named effector-triggered immunity (ETI). ETI needs to be tightly controlled both positively and negatively to enable normal plant growth because constitutively activated defense responses are detrimental to the host. In previous work, we reported that mutations in SUPPRESSOR OF rps4-RLD1 (SRFR1), identified in a suppressor screen, reactivated EDS1-dependent ETI to Pseudomonas syringae pv. tomato (Pto) DC3000. Besides, mutations in SRFR1 boosted defense responses to the generalist chewing insect Spodoptera exigua and the sugar beet cyst nematode Heterodera schachtii. Here, we show that mutations in SRFR1 enhance susceptibility to the fungal necrotrophs Fusarium oxysporum f. sp. lycopersici (FOL) and Botrytis cinerea in Arabidopsis. To translate knowledge obtained in AtSRFR1 research to crops, we generated SlSRFR1 alleles in tomato using a CRISPR/Cas9 system. Interestingly, slsrfr1 mutants increased expression of SA-pathway defense genes and enhanced resistance to Pto DC3000. In contrast, slsrfr1 mutants elevated susceptibility to FOL. Together, these data suggest that SRFR1 is functionally conserved in both Arabidopsis and tomato and functions antagonistically as a negative regulator to (hemi-) biotrophic pathogens and a positive regulator to necrotrophic pathogens.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/imunologia , Arabidopsis/microbiologia , Botrytis/fisiologia , Resistência à Doença/imunologia , Fusarium/fisiologia , Imunidade Vegetal , Alelos , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Sequência de Bases , Sistemas CRISPR-Cas/genética , Resistência à Doença/genética , Edição de Genes , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Vetores Genéticos/metabolismo , Lycopersicon esculentum/genética , Mutação/genética , Imunidade Vegetal/genética , Plasmídeos/genética
2.
Sheng Li Xue Bao ; 73(3): 482-490, 2021 Jun 25.
Artigo em Chinês | MEDLINE | ID: mdl-34230949

RESUMO

S100 calcium binding protein A9 (S100A9) is involved in a variety of biological processes such as inflammation and tumor cell migration and invasion regulation. The purpose of this study was to construct S100A9 gene-edited mice by using CRISPR/Cas9 technology, thereby providing an animal model for exploring the biological functions of this gene. According to the S100A9 gene sequence, the single-stranded small guide RNA (sgRNA) targeting exons 2 and 3 was transcribed in vitro, and a mixture of Cas9 mRNA and candidate sgRNA was injected into mouse fertilized eggs by microinjection. Early embryos were obtained and transferred to surrogate mice, and F0 mice were obtained and identified by PCR identification and gene sequencing. F0 mice were further mated with wild-type C57BL/6 mice to obtain F1 heterozygous mice, and then homozygous offspring were obtained through F1 mice self-crossing. Real-time PCR, Western blot and immunohistochemistry (IHC) were used to verify the expression and distribution of S100A9. In order to observe the pathological changes of mouse lung tissue using HE staining, an allergic asthma model was induced by ovalbumin from chicken egg white (OVA). The results showed that the 2 492 bp of exons 2, 3 of the S100A9 gene was successfully knocked out, and S100A9-/- mice with stable inheritance were obtained. Furthermore, it was found that S100A9 gene was highly expressed in the lung and spleen of wild-type mice. The expression of S100A9 mRNA and protein was not detected in the lung and spleen of S100A9-/- mice. However, compared with wild-type mice, the lungs of S100A9-/- mice showed a significantly worse inflammatory phenotype, and the proportion of eosinophils in bronchoalveolar lavage fluid (BALF) was significantly increased in response to the treatment of OVA. These results suggest we have successfully constructed a new strain of S100A9-/- mice, and preliminarily confirmed that the lack of S100A9 function can aggravate airway inflammation in asthmatic mice, providing a new mouse model for further study of S100A9 gene function.


Assuntos
Sistemas CRISPR-Cas , Marcação de Genes , Animais , Líquido da Lavagem Broncoalveolar , Sistemas CRISPR-Cas/genética , Calgranulina B , Modelos Animais de Doenças , Técnicas de Inativação de Genes , Pulmão , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Ovalbumina , Fenótipo
3.
Yi Chuan ; 43(7): 704-714, 2021 Jul 20.
Artigo em Inglês | MEDLINE | ID: mdl-34284985

RESUMO

Mice are the most widely used model organism for the study of gene functions and disease mechanisms through the generation of gene-modified mice. Since the 1980s, different genetic manipulation technologies have been developed to reveal gene functions in vivo, including homologous recombination strategies mediated by embryonic stem cells, transgenic strategies mediated by gametes, and the latest genetic modification strategies based on CRISPR/Cas9 technology. Semi-cloning technology mediated by "artificial spermatids" (androgenetic haploid embryonic stem cells, also termed sperm-like stem cells) is developed by Chinese scientists in 2012. In combination with CRISPR/Cas9, semi-cloning technology enables one-step generation of gene-modified mice through injection of "artificial spermatids" with specific gene modifications into oocytes. It has the characteristics of short construction cycle, high efficiency, low cost, and high application compatibility. In 2017, the Center for Excellence in Molecular Cell Science (CEMCS) of CAS has launched the genome tagging project (GTP) based on "artificial spermatid"-mediated semi-cloning technology. The ambitious goal of GTP is to tag every protein in mice and construct a unique mouse library that maintains the genome-wide protein-tagging mouse models. Subsequently, the GTP center was established at CEMCS to pursue the project. GTP center developed strategies to generate protein-tagging cells and mice. Briefly, a tag sequence is precisely inserted in a specific protein- coding gene endogenously in cultured "artificial spermatids"in vitro to build a cell library, in which, each cell line carrying a specific protein tag. The tagged cells could be further used as a sperm replacement to produce tagged mice in one step upon injection into oocytes. The tagged mouse library enables global analysis of protein expression, localization, and complexes using standard tag-based assays in vivo. By April 2021, the GTP center has generated 1532 tagged cell lines, 277 of which have been successfully used to produce tagged mice through oocyte injection. A total of 242 tagged mouse strains have been distributed to 66 research teams in 32 research institutions of 15 districts in 3 countries. The database of tagging product resources has been established and released regularly on the GTP website for scientists to inquire and order. Later, more information about GTP products, such as mouse breeding, protein tissue expression map, published literature, etc., will also be successively published on the GTP website. The GTP center will provide a standardized platform for protein function research, which may dramatically promote the development of life science and clinical transformation.


Assuntos
Sistemas CRISPR-Cas , Edição de Genes , Animais , Animais Geneticamente Modificados , Sistemas CRISPR-Cas/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Genoma , Masculino , Camundongos
4.
Yi Chuan ; 43(6): 580-600, 2021 Jun 20.
Artigo em Inglês | MEDLINE | ID: mdl-34284989

RESUMO

Genetic modification technologies can be used for modifying animal genome to express exogenous genes or affect the function of endogenous genes. In animal breeding, genetic modification technologies allow the rapid generation of germplasms with beneficial traits. It includes traditional genetic modification, virus or sperm carrier-mediated genetic modification and nuclease-mediated genome editing, especially the CRISPR/Cas9, one of the artificial nuclease genome editing technologies, have been applied in genome editing in many domestic animals including sheep (Ovis aries). Compared with conventional strategies used for animal breeding, there is great value for sheep breeding improvement by using genome editing technology, which is more effective and timesaving. In this review, we summarize the approaches of genetic modification in sheep and discuss the possibility of molecular design and breeding of sheep by genome editing technologies. We also identify the potential bottlenecks and challenges of these technologies in sheep breeding.


Assuntos
Sistemas CRISPR-Cas , Edição de Genes , Animais , Sistemas CRISPR-Cas/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Melhoramento Vegetal , Ovinos/genética , Tecnologia
5.
Nat Commun ; 12(1): 4219, 2021 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-34244505

RESUMO

Streptococcus pyogenes (Spy) Cas9 has potential as a component of gene therapeutics for incurable diseases. One of its limitations is its large size, which impedes its formulation and delivery in therapeutic applications. Smaller Cas9s are an alternative, but lack robust activity or specificity and frequently recognize longer PAMs. Here, we investigated four uncharacterized, smaller Cas9s and found three employing a "GG" dinucleotide PAM similar to SpyCas9. Protein engineering generated synthetic RNA-guided nucleases (sRGNs) with editing efficiencies and specificities exceeding even SpyCas9 in vitro and in human cell lines on disease-relevant targets. sRGN mRNA lipid nanoparticles displayed manufacturing advantages and high in vivo editing efficiency in the mouse liver. Finally, sRGNs, but not SpyCas9, could be packaged into all-in-one AAV particles with a gRNA and effected robust in vivo editing of non-human primate (NHP) retina photoreceptors. Human gene therapy efforts are expected to benefit from these improved alternatives to existing CRISPR nucleases.


Assuntos
Proteína 9 Associada à CRISPR/genética , Sistemas CRISPR-Cas/genética , Edição de Genes/métodos , RNA Guia/genética , Staphylococcus/enzimologia , Animais , Proteína 9 Associada à CRISPR/isolamento & purificação , Linhagem Celular Tumoral , Modelos Animais de Doenças , Terapia Genética/métodos , Vetores Genéticos/administração & dosagem , Vetores Genéticos/genética , Células HEK293 , Humanos , Macaca fascicularis , Masculino , Camundongos , Parvovirinae/genética , Engenharia de Proteínas , Ribonucleases , Staphylococcus/genética , Especificidade por Substrato , Síndromes de Usher/genética , Síndromes de Usher/terapia
6.
Nat Commun ; 12(1): 4203, 2021 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-34244519

RESUMO

Alternative splicing generates differing RNA isoforms that govern phenotypic complexity of eukaryotes. Its malfunction underlies many diseases, including cancer and cardiovascular diseases. Comparative analysis of RNA isoforms at the genome-wide scale has been difficult. Here, we establish an experimental and computational pipeline that performs de novo transcript annotation and accurately quantifies transcript isoforms from cDNA sequences with a full-length isoform detection accuracy of 97.6%. We generate a searchable, quantitative human transcriptome annotation with 31,025 known and 5,740 novel transcript isoforms ( http://steinmetzlab.embl.de/iBrowser/ ). By analyzing the isoforms in the presence of RNA Binding Motif Protein 20 (RBM20) mutations associated with aggressive dilated cardiomyopathy (DCM), we identify 121 differentially expressed transcript isoforms in 107 cardiac genes. Our approach enables quantitative dissection of complex transcript architecture instead of mere identification of inclusion or exclusion of individual exons, as exemplified by the discovery of IMMT isoforms mis-spliced by RBM20 mutations. Thereby we achieve a path to direct differential expression testing independent of an existing annotation of transcript isoforms, providing more immediate biological interpretation and higher resolution transcriptome comparisons.


Assuntos
Processamento Alternativo , Cardiomiopatia Dilatada/genética , Miócitos Cardíacos/patologia , Proteínas de Ligação a RNA/genética , RNA-Seq/métodos , Sistemas CRISPR-Cas/genética , Cardiomiopatia Dilatada/patologia , Diferenciação Celular/genética , Linhagem Celular , Estudos de Viabilidade , Edição de Genes , Humanos , Células-Tronco Pluripotentes Induzidas , Proteínas Mitocondriais/genética , Anotação de Sequência Molecular , Proteínas Musculares/genética , Mutação , Isoformas de RNA/genética , RNA Guia/genética
7.
BMC Genomics ; 22(1): 563, 2021 Jul 22.
Artigo em Inglês | MEDLINE | ID: mdl-34294050

RESUMO

BACKGROUND: New breeding technologies (NBT) using CRISPR/Cas9-induced homology directed repair (HDR) has the potential to expedite genetic improvement in aquaculture. The long generation time in Atlantic salmon makes breeding an unattractive solution to obtain homozygous mutants and improving the rates of perfect HDR in founder (F0) fish is thus required. Genome editing can represent small DNA changes down to single nucleotide replacements (SNR). This enables edits such as premature stop codons or single amino acid changes and may be used to obtain fish with traits favorable to aquaculture, e.g. disease resistance. A method for SNR has not yet been demonstrated in salmon. RESULTS: Using CRISPR/Cas9 and asymmetrical ODNs, we were able to perform precise SNR and introduce a premature stop codon in dnd in F0 salmon. Deep sequencing demonstrated up to 59.2% efficiency in single embryos. In addition, using the same asymmetrical ODN design, we inserted a FLAG element into slc45a2 and dnd, showing high individual perfect HDR efficiencies (up to 36.7 and 32.7%, respectively). CONCLUSIONS: In this work, we demonstrate that precise SNR and knock-in (KI) can be performed in F0 salmon embryos using asymmetrical oligonucleotide (ODN) donors. We suggest that HDR-induced SNR can be applied as a powerful NBT, allowing efficient introgression of favorable alleles and bypassing challenges associated with traditional selective breeding.


Assuntos
Sistemas CRISPR-Cas , Salmo salar , Alelos , Animais , Sistemas CRISPR-Cas/genética , Edição de Genes , Nucleotídeos , Oligonucleotídeos , Salmo salar/genética
8.
Talanta ; 233: 122591, 2021 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-34215080

RESUMO

The existing CRISPR-mediated diagnostic tests require a two-step procedure (DNA or RNA amplification followed by CRISPR-mediated sequence-specific detection) for nucleic acid detection, which increases complexity and the risk of sample cross-contamination. Here, we report a new CRISPR-mediated test, called CRISPR-top (CRISPR-mediated testing in one-pot), which integrates simultaneous target pre-amplification with CRISPR/cas12b-mediated detection into a one-pot reaction mixture, performed at a constant temperature. The novel CRISPR-top assay was applied to detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent for COVID-19 (coronavirus disease 2019). COVID-19 CRISPR-top targets the ORF1ab (opening reading frame 1a/b) and NP (nucleoprotein) genes of SARS-CoV-2, and operates at 59 °C for 40 min with minimal instrument. The COVID-19 CRISPR-top assay can return results within 60-min and is easily interpreted by visual fluorescence or lateral flow readouts. The analytical limit of detection (LoD) for COVID-19 CRISPR-top is 10 copies (for each detection target) per reaction with no cross-reactivity observed from non-SARS-CoV-2 templates. Among clinically collected non-COVID-19 samples, the assay's specificity was 100% (80/80 oropharynx swab samples). Among 52 COVID-19 positive clinical samples collected, the COVID-19 CRISPR-top assay yielded 38 (73.1%) positive results using fluorescence readout and 35 (67.3%) positive results with lateral-flow readout. These diagnostic results were similar to those obtained using RT-PCR (34 positive (65.4%)). These data indicate that COVID-19 CRISPR-top is a simple, rapid, accurate and highly sensitive method for SARS-CoV-2 detection which can be used in the clinic, field laboratories and primary care facilities in resource-challenged settings.


Assuntos
COVID-19 , Sistemas CRISPR-Cas/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Humanos , Técnicas de Amplificação de Ácido Nucleico , RNA Viral/genética , SARS-CoV-2 , Sensibilidade e Especificidade
9.
Sheng Wu Gong Cheng Xue Bao ; 37(6): 2116-2126, 2021 Jun 25.
Artigo em Chinês | MEDLINE | ID: mdl-34227298

RESUMO

Carrimycin (CAM) is a new antibiotics with isovalerylspiramycins (ISP) as its major components. It is produced by Streptomyces spiramyceticus integrated with a heterogenous 4″-O-isovaleryltransferase gene (ist). However, the present CAM producing strain carries two resistant gene markers, which makes it difficult for further genetic manipulation. In addition, isovalerylation of spiramycin (SP) could be of low efficiency as the ist gene is located far from the SP biosynthesis gene cluster. In this study, ist and its positive regulatory gene acyB2 were inserted into the downstream of orf54 gene neighboring to SP biosynthetic gene cluster in Streptomyces spiramyceticus 1941 by using the CRISPR-Cas9 technique. Two new markerless CAM producing strains, 54IA-1 and 54IA-2, were obtained from the homologous recombination and plasmid drop-out. Interestingly, the yield of ISP in strain 54IA-2 was much higher than that in strain 54IA-1. Quantitative real-time PCR assay showed that the ist, acyB2 and some genes associated with SP biosynthesis exhibited higher expression levels in strain 54IA-2. Subsequently, strain 54IA-2 was subjected to rifampicin (RFP) resistance selection for obtaining high-yield CAM mutants by ribosome engineering. The yield of ISP in mutants resistant to 40 µg/mL RFP increased significantly, with the highest up to 842.9 µg/mL, which was about 6 times higher than that of strain 54IA-2. Analysis of the sequences of the rpoB gene of these 7 mutants revealed that the serine at position 576 was mutated to alanine existed in each sequenced mutant. Among the mutants carrying other missense mutations, strain RFP40-6-8 which carries a mutation of glutamine (424) to leucine showed the highest yield of ISP. In conclusion, two markerless novel CAM producing strains, 54IA-1 and 54IA-2, were successfully developed by using CRISPR-Cas9 technique. Furthermore, a novel CAM high-yielding strain RFP40-6-8 was obtained through ribosome engineering. This study thus demonstrated a useful combinatory approach for improving the production of CAM.


Assuntos
Espiramicina , Streptomyces , Sistemas CRISPR-Cas/genética , Engenharia Genética , Ribossomos , Streptomyces/genética
10.
Talanta ; 232: 122415, 2021 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-34074403

RESUMO

Low abundance gene-PIK3CAH1047R mutation detection is crucial for the clinical diagnosis and treatment of breast cancer. Here, a fluorescent biosensor which combines cascaded strand displacement amplification (C-SDA) and trans-cleavage ability of CRISPR/Cas12a was established to ultra-sensitively detect gene-PIK3CAH1047R mutation. The mutated gene-PIK3CAH1047R can combine with complementary sequence to form an intact recognition site for endonuclease FspI. Mediated by FspI, it breaks at the mutation site to produce DNA fragment to trigger SDA or C-SDA. Then, the fluorescent biosensors based on SDA-CRISPR/Cas12a or C-SDA-CRISPR/Cas12a were constructed. Compared with biosensor based on SDA-CRISPR/Cas12a (5 pM), the minimum detection of the biosensor based on C-SDA-CRISPR/Cas12a is reduced two orders of magnitude (50 fM). In range of 0.001%-50%, we achieved the ultrasensitive detection of gene-PIK3CAH1047R mutation low to 0.001%. Besides, the proposed biosensor works well in human serum samples, showing its application potential in low-abundance gene-PIK3CAH1047R mutation detection.


Assuntos
Técnicas Biossensoriais , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Sistemas CRISPR-Cas/genética , Classe I de Fosfatidilinositol 3-Quinases/genética , Humanos , Mutação
11.
Methods Mol Biol ; 2298: 399-414, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34085257

RESUMO

N6-methyladenosine (m6A) is a major epitranscriptomic mark exerting crucial diverse roles in RNA metabolisms, including RNA stability, mRNA translation, and RNA structural rearrangement. m6A modifications at different RNA regions may have distinct molecular effects. Here, we describe a CRISPR-Cas9-based approach that enables targeted m6A addition or removal on endogenous RNA molecules without altering the nucleotide sequence. By fusing a catalytically inactive Cas9 with engineered m6A modification enzymes, the programmable m6A editors are capable of achieving RNA methylation and demethylation at desired sites, facilitating dissection of regional effects of m6A and diversifying the toolkits for RNA manipulation.


Assuntos
Adenosina/análogos & derivados , Sistemas CRISPR-Cas/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , RNA/genética , Adenosina/genética , Sequência de Bases/genética , Linhagem Celular Tumoral , Edição de Genes/métodos , Células HeLa , Humanos , Metilação , RNA Mensageiro/genética
12.
Int J Mol Sci ; 22(10)2021 May 18.
Artigo em Inglês | MEDLINE | ID: mdl-34069872

RESUMO

Loss-of-function mutations in the synaptosomal-associated protein 29 (SNAP29) lead to the rare autosomal recessive neurocutaneous cerebral dysgenesis, neuropathy, ichthyosis, and keratoderma (CEDNIK) syndrome. SNAP29 is a soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein. So far, it has been shown to be involved in membrane fusion, epidermal differentiation, formation of primary cilia, and autophagy. Recently, we reported the successful generation of two mouse models for the human CEDNIK syndrome. The aim of this investigation was the generation of a CRISPR/Cas9-mediated SNAP29 knockout (KO) in an immortalized human cell line to further investigate the role of SNAP29 in cellular homeostasis and signaling in humans independently of animal models. Comparison of different methods of delivery for CRISPR/Cas9 plasmids into the cell revealed that lentiviral transduction is more efficient than transfection methods. Here, we reported to the best of our knowledge the first successful generation of a CRISPR/Cas9-mediated SNAP29 KO in immortalized human MRC5Vi fibroblasts (c.169_196delinsTTCGT) via lentiviral transduction.


Assuntos
Fibroblastos/metabolismo , Técnicas de Inativação de Genes/métodos , Proteínas Qb-SNARE/genética , Proteínas Qc-SNARE/genética , Animais , Autofagia/genética , Sistemas CRISPR-Cas/genética , Diferenciação Celular/genética , Linhagem Celular , Fibroblastos/fisiologia , Humanos , Ceratodermia Palmar e Plantar/genética , Fusão de Membrana/genética , Mutação/genética , Síndromes Neurocutâneas/genética , Proteínas Qb-SNARE/metabolismo , Proteínas Qc-SNARE/metabolismo , Proteínas SNARE/genética , Proteínas SNARE/metabolismo
13.
Stem Cell Res ; 53: 102346, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-34087983

RESUMO

Spinocerebellar ataxia type 12 (SCA12) is caused by a CAG expansion mutation in PPP2R2B, a gene encoding a brain-specific regulatory unit of protein phosphatase 2A (PP2A); while normal alleles carry 4 to 31 triplets, the disease alleles carry 43 to 78 triplets. Here, by CRISPR/Cas9 genome editing, we have generated a human homozygous SCA12 iPSC line with 69 and 72 triplets for each allele. The homozygous SCA12 iPSCs have normal karyotype, express pluripotency markers and are able to differentiate into the three germ layers.


Assuntos
Células-Tronco Pluripotentes Induzidas , Ataxias Espinocerebelares , Sistemas CRISPR-Cas/genética , Edição de Genes , Homozigoto , Humanos , Mutação/genética , Ataxias Espinocerebelares/genética
14.
Stem Cell Res ; 53: 102350, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-34087984

RESUMO

Human ELTD1 (Epidermal growth factor, latrophilin and seven-transmembrane domain-containing 1), an orphan G-protein-coupled receptor (GPCR) belonging to the adhesion GPCR family, has been reported as a novel regulator of angiogenesis and a potential anti-cancer therapeutic target. However, little is known about the function of ELTD1, especially its undiscovered ligands. In this experiment, an ELTD1 homozygous knockout human embryonic stem cell line, FAHZUe001-A, was generated by the iCRISPR/Cas9 system to achieve a deeper understanding of ELTD1. The FAHZUe001-A was confirmed with normal karyotype, typical undifferentiated morphology, pluripotency and trilineage differentiation potential in vitro.


Assuntos
Células-Tronco Embrionárias Humanas , Neoplasias , Sistemas CRISPR-Cas/genética , Fator de Crescimento Epidérmico , Humanos , Neovascularização Patológica
15.
Stem Cell Res ; 53: 102360, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-34087989

RESUMO

Adult progeria Werner syndrome (WS), a rare autosomal recessive disorder, is characterized by accelerated aging symptoms after puberty. The causative gene, WRN, is a member of the RecQ DNA helicase family and is predominantly involved in DNA replication, repair, and telomere maintenance. Here, we report the generation of iPS cells from a patient with WS and correction of the WRN gene by the CRISPR/Cas9-mediated method. These iPSC lines would be a valuable resource for deciphering the pathogenesis of WS.


Assuntos
Células-Tronco Pluripotentes Induzidas , Síndrome de Werner , Adulto , Sistemas CRISPR-Cas/genética , Exodesoxirribonucleases/genética , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Síndrome de Werner/genética , Helicase da Síndrome de Werner/genética , Helicase da Síndrome de Werner/metabolismo
16.
Stem Cell Res ; 53: 102362, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-34087991

RESUMO

Fat mass and obesity-associated protein (FTO) is the first protein found to have the activity of N6-methyladenosine (m6A) demethylation. It has been reported that FTO was involved in different physiological and pathological processes, including stem cell differentiation, sex determination, tumorigenesis, and progression. To further understand the exact role of FTO in these processes, we generated a FTO knockout human embryonic stem cell (hESC) line by CRISPR/Cas9 mediated gene editing method. This cell line maintained normal karyotype, pluripotency, and trilineage differentiation potential, which are considered as a model for function studies of the FTO protein in hESC self-renewal and differentiation.


Assuntos
Sistemas CRISPR-Cas , Células-Tronco Embrionárias Humanas , Dioxigenase FTO Dependente de alfa-Cetoglutarato/genética , Sistemas CRISPR-Cas/genética , Linhagem Celular , Edição de Genes , Técnicas de Inativação de Genes , Humanos
17.
Stem Cell Res ; 53: 102363, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-34087992

RESUMO

ISL1 encodes a member of the LIM/homeodomain family of transcription factors. This encoded protein plays central roles in the development of motor neuron, pancreas, and secondary heart field. Here we generated heterozygous fluorescent reporters of the ISL1 gene in human induced pluripotent stem cells (hiPSCs). CRISPR/Cas9 genome editing technology was employed to knock-in 2A-tdTomato and EF1 alpha promoter-driven Bleomycin resistance gene to the translational ISL1 C-terminal region. The resulting ISL1-TEZ lines showed tdTomato fluorescence upon motor neuron differentiation. These reporter iPSC lines provide opportunity for monitoring and purifying these related cell lineages.


Assuntos
Edição de Genes , Células-Tronco Pluripotentes Induzidas , Sistemas CRISPR-Cas/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Humanos , Proteínas Luminescentes
18.
Sci Transl Med ; 13(598)2021 06 16.
Artigo em Inglês | MEDLINE | ID: mdl-34135108

RESUMO

Sickle cell disease (SCD) is the most common serious monogenic disease with 300,000 births annually worldwide. SCD is an autosomal recessive disease resulting from a single point mutation in codon six of the ß-globin gene (HBB). Ex vivo ß-globin gene correction in autologous patient-derived hematopoietic stem and progenitor cells (HSPCs) may potentially provide a curative treatment for SCD. We previously developed a CRISPR-Cas9 gene targeting strategy that uses high-fidelity Cas9 precomplexed with chemically modified guide RNAs to induce recombinant adeno-associated virus serotype 6 (rAAV6)-mediated HBB gene correction of the SCD-causing mutation in HSPCs. Here, we demonstrate the preclinical feasibility, efficacy, and toxicology of HBB gene correction in plerixafor-mobilized CD34+ cells from healthy and SCD patient donors (gcHBB-SCD). We achieved up to 60% HBB allelic correction in clinical-scale gcHBB-SCD manufacturing. After transplant into immunodeficient NSG mice, 20% gene correction was achieved with multilineage engraftment. The long-term safety, tumorigenicity, and toxicology study demonstrated no evidence of abnormal hematopoiesis, genotoxicity, or tumorigenicity from the engrafted gcHBB-SCD drug product. Together, these preclinical data support the safety, efficacy, and reproducibility of this gene correction strategy for initiation of a phase 1/2 clinical trial in patients with SCD.


Assuntos
Anemia Falciforme , Compostos Heterocíclicos , Anemia Falciforme/genética , Anemia Falciforme/terapia , Animais , Sistemas CRISPR-Cas/genética , Edição de Genes , Mobilização de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas , Humanos , Camundongos , Reprodutibilidade dos Testes , Globinas beta/genética
19.
Zhejiang Da Xue Xue Bao Yi Xue Ban ; 50(2): 229-238, 2021 04 25.
Artigo em Inglês | MEDLINE | ID: mdl-34137224

RESUMO

To establish a rabbit model of proprotein convertase subtilisin/kexin type9 () point mutation with CRISPR/Cas9 gene editing technique. According to the PubMed gene protein data, the PCSK9 protein functional regions of human and rabbit were analyzed by Blast. The 386S (Ser) amino acid functional region of human gene was homologous to the 485S of rabbit gene. Three small guide RNAs and one single-stranded donor oligonucleotide were designed according to the 485S base substitution position and sequence analysis of rabbit gene. The synthetic small guide RNAs, Cas9 mRNA and single-stranded donor oligonucleotide were co-injected into the cytoplasm of rabbit fertilized eggs and the embryos were transferred into the pregnant rabbits. PCR, TA cloning and off-target analysis were performed on the F0 rabbits to identify whether the PCSK9 mutation was successful. Fifteen F0 rabbits were obtained. The sequencing results showed that one of them was PCSK9 point mutation homozygote and two of them were PCSK9 point mutation heterozygotes, and the mutation could be stably inherited. The rabbit model of PCSK9 point mutation was successfully constructed by CRISPR/Cas9 technique, which provides an animal model for exploring the molecular mechanism of impaired PCSK9 function and developing reliable and effective diagnosis and treatment measures.


Assuntos
Sistemas CRISPR-Cas , Pró-Proteína Convertase 9 , Animais , Sistemas CRISPR-Cas/genética , Mutação , Mutação Puntual , Pró-Proteína Convertase 9/genética , Pró-Proteína Convertase 9/metabolismo , Coelhos
20.
Bone ; 151: 116036, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34118444

RESUMO

Multiple actions of extracellular Pi on the skeletal cells are likely to be partly mediated by type III sodium/phosphate (Na+/Pi) cotransporters Pit1 and Pit2, although the details are not fully understood. In the current study, to determine the roles of Pit1 and Pit2 in osteoblasts, we generated Pit1-knockout (KO) and Pit2-KO osteoblastic cells by applying CRISPR/Cas9 genome editing to an osteoblastic cell line MC3T3-E1 subclone 4. The extracellular Pi level was increased in the Pit1-KO and Pit2-KO clones due to the reduced Pi uptake. Interestingly, in vitro mineralization was accelerated in the Pit1-KO and Pit2-KO clones, although the induction of the expression of osteogenic marker genes was suppressed. In the cells before mineralization, extracellular levels of pyrophosphate (PPi) and adenosine triphosphate (ATP) were increased in the Pit1-KO and Pit2-KO clones, which might be attributable to the reduced expression and activity of tissue-nonspecific alkaline phosphatase (TNSALP). A 24-h treatment with high Pi reduced the expression and activity of TNSALP, suggesting that the suppression of TNSALP in the Pit1-KO and Pit2-KO clones was caused by the increased availability of extracellular Pi. Lentiviral gene transfer of Pit1 and Pit2 restored the changes observed in Pit1-KO and Pit2-KO clones, respectively. The expressions of P2Y2 and P2X7 which encode receptors for extracellular ATP were altered in the Pit1-KO and Pit2-KO clones, suggesting an influence on purinergic signaling. In mineralized cells after long-term culture, intracellular levels of PPi and ATP were higher in the Pit1-KO and Pit2-KO clones. Taken together, ablation of Pit1 or Pit2 in this osteoblastic cell model led to accelerated mineralization, suppressed TNSALP and altered the levels of extracellular and intracellular PPi and ATP, which might be partly mediated by changes in the availability of extracellular Pi.


Assuntos
Sistemas CRISPR-Cas , Proteínas Cotransportadoras de Sódio-Fosfato Tipo III , Transporte Biológico , Sistemas CRISPR-Cas/genética , Linhagem Celular , Expressão Gênica , Proteínas Cotransportadoras de Sódio-Fosfato Tipo III/genética , Proteínas Cotransportadoras de Sódio-Fosfato Tipo III/metabolismo
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