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1.
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
2.
Mol Ther Nucleic Acids ; 19: 775-789, 2020 Mar 06.
Artigo em Inglês | MEDLINE | ID: mdl-31955009

RESUMO

CRISPR/Cas9-mediated homology-directed repair (HDR) can be leveraged to precisely engineer mammalian genomes. However, the inherently low efficiency of HDR often hampers to identify the desired modified cells. Here, we developed a novel universal surrogate reporter system that efficiently enriches for genetically modified cells arising from CRISPR/Cas9-induced HDR events (namely, the "HDR-USR" system). This episomally based reporter can be self-cleaved and self-repaired via HDR to create a functional puromycin selection cassette without compromising genome integrity. Co-transfection of the HDR-USR system into host cells and transient puromycin selection efficiently achieves enrichment of HDR-modified cells. We tested the system for precision point mutation at 16 loci in different human cell lines and one locus in two rodent cell lines. This system exhibited dramatic improvements in HDR efficiency at a single locus (up to 20.7-fold) and two loci at once (42% editing efficiency compared to zero in the control), as well as greatly improved knockin efficiency (8.9-fold) and biallelic deletion (35.9-fold) at test loci. Further increases were achieved by co-expression of yeast Rad52 and linear single-/double-stranded DNA donors. Taken together, our HDR-USR system provides a simple, robust and efficient surrogate reporter for the enrichment of CRISPR/Cas9-induced HDR-based precision genome editing across various targeting loci in different cell lines.

3.
Front Genet ; 10: 347, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31057603

RESUMO

The SNP within intron 3 of the porcine IGF2 gene (G3072A) plays an important role for muscle growth and fat deposition in pigs. In this study, the StCas9 derived from Streptococcus thermophilus together with the Drosha-mediated sgRNA-shRNA structure were combined to boost the G to A base editing on the IGF2 SNP site, which we called "SNP editing." The codon-humanized StCas9 as we previously reported was firstly compared with the prevalently used SpCas9 derived from Streptococcus pyogenes using our idiomatic surrogate report assay, and the StCas9 demonstrated a comparable targeting activity. On the other hand, by combining shRNA with sgRNA, simultaneous gene silencing and genome targeting can be achieved. Thus, the novel IGF2.sgRNA-LIG4.shRNA-IGF2.sgRNA structure was constructed to enhance the sgRNA/Cas9-mediated HDR-based IGF2 SNP editing by silencing the LIG4 gene, which is a key molecule of the HDR's competitive NHEJ pathway. The sgRNA-shRNA/StCas9 all-in-one expression vector and the IGF2.sgRNA/StCas9 as control were separately used to transfect porcine PK15 cells together with an ssODNs donor for the IGF2 SNP editing. The editing events were detected by the RFLP assay, Sanger sequencing as well as Deep-sequencing, and the Deep-sequencing results finally demonstrated a significant higher HDR-based editing efficiency (16.38%) for our sgRNA-shRNA/StCas9 strategy. In short, we achieved effective IGF2 SNP editing by using the combined sgRNA-shRNA/StCas9 strategy, which will facilitate the further production of base-edited animals and perhaps extend for the gene therapy for the base correction of some genetic diseases.

4.
PLoS One ; 11(8): e0160989, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27518717

RESUMO

Matrix metalloproteinase-9 (MMP-9) is a zinc-dependent enzyme, and plays a crucial role in extracellular matrix degeneration, inflammation and tissue remodeling. However, the relationship between MMP-9 and somatic cell count (SCC) in goat milk and the role of MMP-9 in the regulation of mastitis are still unknown. In this study, we found MMP-9 was predominantly expressed in the spleen, intestine and mammary gland. The SCC in goat milk was positively correlated with MMP-9 expression, and staphylococcus aureus could markedly increase MMP-9 expression in goat mammary epithelial cells (GMEC) in dosage and time dependent manner. We also demonstrated that SB-3CT, an inhibitor of MMP-9, promoted apoptosis and inhibited proliferation in GMEC. Thus, MMP-9 may emerge as an easily measurable and sensitive parameter that reflects the number of somatic cells present in milk and a regulatory factor of apoptosis in GMEC.


Assuntos
Indústria de Laticínios , Cabras , Glândulas Mamárias Animais/patologia , Mastite/enzimologia , Mastite/patologia , Metaloproteinase 9 da Matriz/metabolismo , Animais , Apoptose , Contagem de Células , Sobrevivência Celular , Feminino , Regulação Enzimológica da Expressão Gênica , Glândulas Mamárias Animais/microbiologia , Mastite/metabolismo , Mastite/microbiologia , Metaloproteinase 9 da Matriz/genética , Especificidade de Órgãos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Staphylococcus aureus/fisiologia
5.
Mol Biol Rep ; 42(1): 233-43, 2015 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25266236

RESUMO

Normal mammary gland epithelial cells and breast cancer cells express the calcium-sensing receptor (CaSR), which is the master regulator of systemic calcium metabolism. During lactation, activation of the CaSR in mammary epithelial cells downregulates parathyroid hormone-related protein (PTHrP) levels in milk and in the circulation, and increases calcium transport into milk. However, very little information is available on the role of CaSR in goat mammary gland epithelial cells (GMECs) apoptosis. In this investigation, the full-length cDNA of CaSR from Xinong Saanen dairy goats was cloned, which contains an open-reading frame of 3,258 bp encoding 1,085 amino acids with a predicted molecular weight of 121.0 kDa and an isoelectric point of 5.65. The amino acid sequence is highly homologous with sheep, and the goat CaSR gene is mapped to chromosome 1. Quantitative real-time PCR suggested that CaSR was predominantly expressed in the heart, kidney and mammary gland. Then, we found the stimulation of CaSR with its activator gadolinium chloride (GdCl3) contributed to increase CaSR mRNA levels in GMECs and simultaneously promoted cell apoptosis, and these effects were abrogated partially by NPS2390 which is an inhibitor of CaSR. We also demonstrated that Ca(2+) increased CaSR mRNA levels and induced GMECs apoptosis and restrained cell proliferation. In contrast, PTHrP overexpression protected GMECs from calcium-induced apoptosis, and promoted cell proliferation. In conclusion, these results suggest that PTHrP overexpression protects GMECs from CaSR activation-induced apoptosis.


Assuntos
Apoptose , Citoproteção , Células Epiteliais/metabolismo , Cabras/metabolismo , Glândulas Mamárias Animais/citologia , Proteína Relacionada ao Hormônio Paratireóideo/metabolismo , Receptores de Detecção de Cálcio/metabolismo , Sequência de Aminoácidos , Animais , Apoptose/efeitos dos fármacos , Sequência de Bases , Proliferação de Células/efeitos dos fármacos , Citoproteção/efeitos dos fármacos , Citoproteção/genética , Células Epiteliais/efeitos dos fármacos , Feminino , Gadolínio/farmacologia , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Dados de Sequência Molecular , Especificidade de Órgãos/genética , Filogenia , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Receptores de Detecção de Cálcio/química , Receptores de Detecção de Cálcio/genética
6.
Gene ; 551(2): 279-89, 2014 Nov 10.
Artigo em Inglês | MEDLINE | ID: mdl-25194899

RESUMO

The signal transducer and activator of transcription 5a (Stat5a) modulates genes involved in proliferation and survival and plays pivotal roles in regulating the function of the mammary gland during pregnancy, lactation, and involution. However, there is little information about the effects of Stat5a on apoptosis of goat mammary gland epithelial cells (GMECs). In addition, parathyroid hormone-related protein (PTHrP) is a key regulator in cellular calcium transport, mammary gland development and breast tumor biology. This study aimed to explore the interaction of Stat5a and PTHrP in GMEC apoptosis. Quantitative real time PCR (qRT-PCR) suggested that Stat5a was predominantly expressed in the mammary gland, lung, liver and spleen of goats. Treating the GMECs with pimozide, an inhibitor of Stat5a that decreases Stat5a tyrosine phosphorylation, increased PTHrP levels in GMECs in a dose-dependent manner and simultaneously promoted apoptosis of the GMECs. We also demonstrated that PTHrP inhibition induced GMEC apoptosis and restrained cell proliferation. In contrast, PTHrP overexpression protected GMECs from pimozide- and calcium-induced apoptosis, and promoted cell proliferation. Furthermore, pimozide and CaCl2 downregulated the antiapoptotic protein Bcl-2 mRNA expression, respectively, and these effects were protected by PTHrP overexpression. Interestingly, we also found that Stat5a suppressed the expression of matrix metalloproteinase 9 (MMP-9) which can induce goat mammary epithelial cell migration, but PTHrP increased MMP-9 mRNA level. Thus, Stat5a may regulate GMEC survival by regulating the expression of PTHrP.


Assuntos
Cabras/genética , Glândulas Mamárias Animais/metabolismo , Proteína Relacionada ao Hormônio Paratireóideo/genética , Pimozida/farmacologia , Fator de Transcrição STAT5/genética , Sequência de Aminoácidos , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , Sequência de Bases , Cálcio/metabolismo , Cálcio/farmacologia , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Células Cultivadas , Antagonistas de Dopamina/farmacologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Feminino , Citometria de Fluxo , Expressão Gênica/efeitos dos fármacos , Perfilação da Expressão Gênica , Cabras/metabolismo , Glândulas Mamárias Animais/citologia , Metaloproteinase 9 da Matriz/genética , Metaloproteinase 9 da Matriz/metabolismo , Dados de Sequência Molecular , Proteína Relacionada ao Hormônio Paratireóideo/metabolismo , Fosforilação/efeitos dos fármacos , Filogenia , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator de Transcrição STAT5/classificação , Fator de Transcrição STAT5/metabolismo
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