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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.
Nat Methods ; 18(5): 499-506, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33941935

RESUMO

Competitive coevolution between microbes and viruses has led to the diversification of CRISPR-Cas defense systems against infectious agents. By analyzing metagenomic terabase datasets, we identified two compact families (775 to 803 amino acids (aa)) of CRISPR-Cas ribonucleases from hypersaline samples, named Cas13X and Cas13Y. We engineered Cas13X.1 (775 aa) for RNA interference experiments in mammalian cell lines. We found Cas13X.1 could tolerate single-nucleotide mismatches in RNA recognition, facilitating prophylactic RNA virus inhibition. Moreover, a minimal RNA base editor, composed of engineered deaminase (385 aa) and truncated Cas13X.1 (445 aa), exhibited robust editing efficiency and high specificity to induce RNA base conversions. Our results suggest that there exist untapped bacterial defense systems in natural microbes that can function efficiently in mammalian cells, and thus potentially are useful for RNA-editing-based research.


Assuntos
Sistemas CRISPR-Cas , Edição de RNA , RNA Bacteriano , Animais , Proteínas de Bactérias , Linhagem Celular , Clonagem Molecular , Bases de Dados de Ácidos Nucleicos , Cães , Humanos , Camundongos , Interferência de RNA
3.
Sci Adv ; 6(29): eaba1773, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32832622

RESUMO

Cytosine base editors (CBEs) enable efficient cytidine-to-thymidine (C-to-T) substitutions at targeted loci without double-stranded breaks. However, current CBEs edit all Cs within their activity windows, generating undesired bystander mutations. In the most challenging circumstance, when a bystander C is adjacent to the targeted C, existing base editors fail to discriminate them and edit both Cs. To improve the precision of CBE, we identified and engineered the human APOBEC3G (A3G) deaminase; when fused to the Cas9 nickase, the resulting A3G-BEs exhibit selective editing of the second C in the 5'-CC-3' motif in human cells. Our A3G-BEs could install a single disease-associated C-to-T substitution with high precision. The percentage of perfectly modified alleles is more than 6000-fold for disease correction and more than 600-fold for disease modeling compared with BE4max. On the basis of the two-cell embryo injection method and RNA sequencing analysis, our A3G-BEs showed minimum genome- and transcriptome-wide off-target effects, achieving high targeting fidelity.

4.
Nat Protoc ; 15(9): 3009-3029, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32796939

RESUMO

Genome editing holds great potential for correcting pathogenic mutations. We developed a method called GOTI (genome-wide off-target analysis by two-cell embryo injection) to detect off-target mutations by editing one blastomere of two-cell mouse embryos using either CRISPR-Cas9 or base editors. GOTI directly compares edited and non-edited cells without the interference of genetic background and thus could detect potential off-target variants with high sensitivity. Notably, the GOTI method was designed to detect potential off-target variants of any genome editing tools by the combination of experimental and computational approaches, which is critical for accurate evaluation of the safety of genome editing tools. Here we provide a detailed protocol for GOTI, including mice mating, two-cell embryo injection, embryonic day 14.5 embryo digestion, fluorescence-activated cell sorting, whole-genome sequencing and data analysis. To enhance the utility of GOTI, we also include a computational workflow called GOTI-seq (https://github.com/sydaileen/GOTI-seq) for the sequencing data analysis, which can generate the final genome-wide off-target variants from raw sequencing data directly. The protocol typically takes 20 d from the mice mating to sequencing and 7 d for sequencing data analysis.


Assuntos
Embrião de Mamíferos/metabolismo , Edição de Genes/métodos , Animais , Feminino , Injeções , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mutação
5.
Nat Methods ; 17(6): 600-604, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32424272

RESUMO

Cytosine base editors (CBEs) offer a powerful tool for correcting point mutations, yet their DNA and RNA off-target activities have caused concerns in biomedical applications. We describe screens of 23 rationally engineered CBE variants, which reveal mutation residues in the predicted DNA-binding site can dramatically decrease the Cas9-independent off-target effects. Furthermore, we obtained a CBE variant-YE1-BE3-FNLS-that retains high on-target editing efficiency while causing extremely low off-target edits and bystander edits.


Assuntos
Proteína 9 Associada à CRISPR/genética , Citosina/metabolismo , DNA/genética , Edição de Genes/métodos , RNA/genética , Sequência de Bases , Sistemas CRISPR-Cas/genética , Células HEK293 , Humanos , Mutação , Mutação Puntual
6.
Science ; 364(6437): 289-292, 2019 04 19.
Artigo em Inglês | MEDLINE | ID: mdl-30819928

RESUMO

Genome editing holds promise for correcting pathogenic mutations. However, it is difficult to determine off-target effects of editing due to single-nucleotide polymorphism in individuals. Here we developed a method named GOTI (genome-wide off-target analysis by two-cell embryo injection) to detect off-target mutations by editing one blastomere of two-cell mouse embryos using either CRISPR-Cas9 or base editors. Comparison of the whole-genome sequences of progeny cells of edited and nonedited blastomeres at embryonic day 14.5 showed that off-target single-nucleotide variants (SNVs) were rare in embryos edited by CRISPR-Cas9 or adenine base editor, with a frequency close to the spontaneous mutation rate. By contrast, cytosine base editing induced SNVs at more than 20-fold higher frequencies, requiring a solution to address its fidelity.


Assuntos
Blastômeros , Proteína 9 Associada à CRISPR , Sistemas CRISPR-Cas , Citosina , Edição de Genes/métodos , Polimorfismo de Nucleotídeo Único , Animais , Análise Mutacional de DNA , Embrião de Mamíferos , Feminino , Estudo de Associação Genômica Ampla , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mutação
7.
Acta Biochim Biophys Sin (Shanghai) ; 50(6): 540-546, 2018 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-29688249

RESUMO

Prolyl hydroxylases (PHD1-3) hydroxylate hypoxia inducible factor α (HIFα), leading to HIFα ubiquitination and degradation. Recent studies indicated that administration of generic inhibitors of PHDs improved mice colitis, suggesting that suppression of PHD activity by these inhibitors may be a potential strategy for the treatment of inflammatory bowel diseases. However, the exact role of each member of PHD family in homeostasis of intestinal epithelium remains elusive. The aim of this work is to study the possible role of PHD2 by using mice with genetic ablation of Phd2 in intestinal epithelial cells (IECs). We found that deletion of PHD2 in IECs did not lead to spontaneous enteritis or colitis in mice. Deletion of PHD2 in IECs did not confer upon mice higher susceptibility to dextran sodium sulfate-induced colitis. Furthermore, in a colitis-associated colon cancer model, the PHD2-conditional knockout mice had similar susceptibility to azoxymethane (AOM)-induced colonic tumorigenesis as control mice did. Our results suggest that PHD2 is dispensable for maintenance of intestinal epithelium homeostasis in mice.


Assuntos
Células Epiteliais/metabolismo , Homeostase/genética , Prolina Dioxigenases do Fator Induzível por Hipóxia/genética , Mucosa Intestinal/metabolismo , Animais , Azoximetano , Linhagem Celular , Linhagem Celular Tumoral , Colite/induzido quimicamente , Colite/genética , Neoplasias do Colo/induzido quimicamente , Neoplasias do Colo/genética , Sulfato de Dextrana , Humanos , Prolina Dioxigenases do Fator Induzível por Hipóxia/metabolismo , Camundongos Knockout , Camundongos Transgênicos
8.
J Biol Chem ; 290(33): 20580-9, 2015 Aug 14.
Artigo em Inglês | MEDLINE | ID: mdl-26124271

RESUMO

Prolyl hydroxylase domain proteins (PHDs) control cellular adaptation to hypoxia. PHDs are found involved in inflammatory bowel disease (IBD); however, the exact role of PHD3, a member of the PHD family, in IBD remains unknown. We show here that PHD3 plays a critical role in maintaining intestinal epithelial barrier function. We found that genetic ablation of Phd3 in intestinal epithelial cells led to spontaneous colitis in mice. Deletion of PHD3 decreases the level of tight junction protein occludin, leading to a failure of intestinal epithelial barrier function. Further studies indicate that PHD3 stabilizes occludin by preventing the interaction between the E3 ligase Itch and occludin, in a hydroxylase-independent manner. Examination of biopsy of human ulcerative colitis patients indicates that PHD3 is decreased with disease severity, indicating that PHD3 down-regulation is associated with progression of this disease. We show that PHD3 protects intestinal epithelial barrier function and reveal a hydroxylase-independent function of PHD3 in stabilizing occludin. These findings may help open avenues for developing a therapeutic strategy for IBD.


Assuntos
Mucosa Intestinal/fisiologia , Ocludina/fisiologia , Pró-Colágeno-Prolina Dioxigenase/fisiologia , Animais , Colite/genética , Colite/prevenção & controle , Deleção de Genes , Células HEK293 , Humanos , Camundongos , Camundongos Transgênicos
9.
J Biol Chem ; 290(24): 15327-36, 2015 Jun 12.
Artigo em Inglês | MEDLINE | ID: mdl-25925952

RESUMO

Intestinal epithelial cells (IECs) have critical roles in maintaining homeostasis of intestinal epithelium. Endoplasmic reticulum (ER) stress is implicated in intestinal epithelium homeostasis and inflammatory bowel disease; however, it remains elusive whether IRE1α, a major sensor of ER stress, is directly involved in these processes. We demonstrate here that genetic ablation of Ire1α in IECs leads to spontaneous colitis in mice. Deletion of IRE1α in IECs results in loss of goblet cells and failure of intestinal epithelial barrier function. IRE1α deficiency induces cell apoptosis through induction of CHOP, the pro-apoptotic protein, and sensitizes cells to lipopolysaccharide, an endotoxin from bacteria. IRE1α deficiency confers upon mice higher susceptibility to chemical-induced colitis. These results suggest that IRE1α functions to maintain the intestinal epithelial homeostasis and plays an important role in defending against inflammation bowel diseases.


Assuntos
Colite/prevenção & controle , Retículo Endoplasmático/metabolismo , Endorribonucleases/fisiologia , Mucosa Intestinal/metabolismo , Proteínas Serina-Treonina Quinases/fisiologia , Animais , Sequência de Bases , Linhagem Celular , Primers do DNA , Endorribonucleases/genética , Homeostase , Camundongos , Camundongos Transgênicos , Reação em Cadeia da Polimerase , Proteínas Serina-Treonina Quinases/genética , RNA Interferente Pequeno/genética
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