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1.
Cell Prolif ; 54(8): e13096, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34240779

RESUMO

OBJECTIVES: PKM1 and PKM2, which are generated from the alternative splicing of PKM gene, play important roles in tumourigenesis and embryonic development as rate-limiting enzymes in glycolytic pathway. However, because of the lack of appropriate techniques, the specific functions of the 2 PKM splicing isoforms have not been clarified endogenously yet. MATERIALS AND METHODS: In this study, we used CRISPR-based base editors to perturbate the endogenous alternative splicing of PKM by introducing mutations into the splicing junction sites in HCT116 cells and zebrafish embryos. Sanger sequencing, agarose gel electrophoresis and targeted deep sequencing assays were utilized for identifying mutation efficiencies and detecting PKM1/2 splicing isoforms. Cell proliferation assays and RNA-seq analysis were performed to describe the effects of perturbation of PKM1/2 splicing in tumour cell growth and zebrafish embryo development. RESULTS: The splicing sites of PKM, a 5' donor site of GT and a 3' acceptor site of AG, were efficiently mutated by cytosine base editor (CBE; BE4max) and adenine base editor (ABE; ABEmax-NG) with guide RNAs (gRNAs) targeting the splicing sites flanking exons 9 and 10 in HCT116 cells and/or zebrafish embryos. The mutations of the 5' donor sites of GT flanking exons 9 or 10 into GC resulted in specific loss of PKM1 or PKM2 expression as well as the increase in PKM2 or PKM1 respectively. Specific loss of PKM1 promoted cell proliferation of HCT116 cells and upregulated the expression of cell cycle regulators related to DNA replication and cell cycle phase transition. In contrast, specific loss of PKM2 suppressed cell growth of HCT116 cells and resulted in growth retardation of zebrafish. Meanwhile, we found that mutation of PKM1/2 splicing sites also perturbated the expression of non-canonical PKM isoforms and produced some novel splicing isoforms. CONCLUSIONS: This work proved that CRISPR-based base editing strategy can be used to disrupt the endogenous alternative splicing of genes of interest to study the function of specific splicing isoforms in vitro and in vivo. It also reminded us to notice some novel or undesirable splicing isoforms by targeting the splicing junction sites using base editors. In sum, we establish a platform to perturbate endogenous RNA splicing for functional investigation or genetic correction of abnormal splicing events in human diseases.


Assuntos
Edição de Genes , Piruvato Quinase/metabolismo , Processamento Alternativo , Animais , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Proliferação de Células , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Regulação para Baixo , Éxons , Feminino , Células HCT116 , Humanos , Mutagênese , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Piruvato Quinase/genética , Regulação para Cima , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo
2.
Mol Ther ; 2021 May 08.
Artigo em Inglês | MEDLINE | ID: mdl-33974999

RESUMO

A couple diagnosed as carriers for lamellar ichthyosis, an autosomal recessive rare disease, encountered two pregnancy losses. Their blood samples showed the same heterozygous c.607C>T mutation in the TGM1 gene. However, we found that about 98.4% of the sperm had mutations, suggesting possible de novo germline mutation. To explore the probability of correcting this mutation, we used two different adenine base editors (ABEs) combined with related truncated single guide RNA (sgRNA) to repair the pathogenic mutation in mutant zygotes. Our results showed that the editing efficiency was 73.8% for ABEmax-NG combined with 20-bp-length sgRNA and 78.7% for Sc-ABEmax combined with 19-bp-length sgRNA. The whole-genome sequencing (WGS) and deep sequencing analysis demonstrated precise DNA editing. This study reveals the possibility of correcting the genetic mutation in embryos with the ABE system.

3.
Nat Commun ; 12(1): 2287, 2021 04 16.
Artigo em Inglês | MEDLINE | ID: mdl-33863894

RESUMO

Both adenine base editors (ABEs) and cytosine base editors (CBEs) have been recently revealed to induce transcriptome-wide RNA off-target editing in a guide RNA-independent manner. Here we construct a reporter system containing E.coli Hokb gene with a tRNA-like motif for robust detection of RNA editing activities as the optimized ABE, ABEmax, induces highly efficient A-to-I (inosine) editing within an E.coli tRNA-like structure. Then, we design mutations to disrupt the potential interaction between TadA and tRNAs in structure-guided principles and find that Arginine 153 (R153) within TadA is essential for deaminating RNAs with core tRNA-like structures. Two ABEmax or mini ABEmax variants (TadA* fused with Cas9n) with deletion of R153 within TadA and/or TadA* (named as del153/del153* and mini del153) are successfully engineered, showing minimized RNA off-targeting, but comparable DNA on-targeting activities. Moreover, R153 deletion in recently reported ABE8e or ABE8s can also largely reduce their RNA off-targeting activities. Taken together, we develop a strategy to generate engineered ABEs (eABEs) with minimized RNA off-targeting activities.


Assuntos
Adenosina Desaminase/genética , Proteína 9 Associada à CRISPR/genética , DNA/genética , Proteínas de Escherichia coli/genética , Edição de Genes/métodos , Adenina/metabolismo , Adenosina Desaminase/metabolismo , Toxinas Bacterianas/genética , Proteína 9 Associada à CRISPR/metabolismo , Linhagem Celular Tumoral , Citosina/metabolismo , DNA/metabolismo , Proteínas de Escherichia coli/metabolismo , Genes Reporter , Células HEK293 , Humanos , Inosina/genética , Inosina/metabolismo , Engenharia de Proteínas , Edição de RNA/genética , RNA de Transferência/genética , RNA de Transferência/metabolismo , RNA-Seq , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo
4.
BMC Biol ; 19(1): 34, 2021 02 18.
Artigo em Inglês | MEDLINE | ID: mdl-33602235

RESUMO

BACKGROUND: Site-specific C>T DNA base editing has been achieved by recruiting cytidine deaminases to the target C using catalytically impaired Cas proteins; the target C is typically located within 5-nt editing window specified by the guide RNAs. The prototypical cytidine base editor BE3, comprising rat APOBEC1 (rA1) fused to nCas9, can indiscriminately deaminate multiple C's within the editing window and also create substantial off-target edits on the transcriptome. A powerful countermeasure for the DNA off-target editing is to replace rA1 with APOBEC proteins which selectively edit C's in the context of specific motifs, as illustrated in eA3A-BE3 which targets TC. However, analogous editors selective for other motifs have not been described. In particular, it has been challenging to target a particular C in C-rich sequences. Here, we sought to confront this challenge and also to overcome the RNA off-target effects seen in BE3. RESULTS: By replacing rA1 with an optimized human A3G (oA3G), we developed oA3G-BE3, which selectively targets CC and CCC and is also free of global off-target effects on the transcriptome. Furthermore, we created oA3G-BE4max, an upgraded version of oA3G-BE3 with robust on-target editing. Finally, we showed that oA3G-BE4max has negligible Cas9-independent off-target effects at the genome. CONCLUSIONS: oA3G-BE4max can edit C(C)C with high efficiency and selectivity, which complements eA3A-editors to broaden the collective editing scope of motif selective editors, thus filling a void in the base editing tool box.


Assuntos
Desaminase APOBEC-3G/genética , Sistemas CRISPR-Cas , Citidina Desaminase/metabolismo , Edição de Genes , RNA Guia
5.
Sci China Life Sci ; 64(1): 1-21, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33165812

RESUMO

The rumen is the hallmark organ of ruminants and hosts a diverse ecosystem of microorganisms that facilitates efficient digestion of plant fibers. We analyzed 897 transcriptomes from three Cetartiodactyla lineages: ruminants, camels and cetaceans, as well as data from ruminant comparative genomics and functional assays to explore the genetic basis of rumen functional innovations. We identified genes with relatively high expression in the rumen, of which many appeared to be recruited from other tissues. These genes show functional enrichment in ketone body metabolism, regulation of microbial community, and epithelium absorption, which are the most prominent biological processes involved in rumen innovations. Several modes of genetic change underlying rumen functional innovations were uncovered, including coding mutations, genes newly evolved, and changes of regulatory elements. We validated that the key ketogenesis rate-limiting gene (HMGCS2) with five ruminant-specific mutations was under positive selection and exhibits higher synthesis activity than those of other mammals. Two newly evolved genes (LYZ1 and DEFB1) are resistant to Gram-positive bacteria and thereby may regulate microbial community equilibrium. Furthermore, we confirmed that the changes of regulatory elements accounted for the majority of rumen gene recruitment. These results greatly improve our understanding of rumen evolution and organ evo-devo in general.

6.
Nat Commun ; 11(1): 6073, 2020 11 27.
Artigo em Inglês | MEDLINE | ID: mdl-33247095

RESUMO

DNA base editors, typically comprising editing enzymes fused to the N-terminus of nCas9, display off-target effects on DNA and/or RNA, which have remained an obstacle to their clinical applications. Off-target edits are typically countered via rationally designed point mutations, but the approach is tedious and not always effective. Here, we report that the off-target effects of both A > G and C > T editors can be dramatically reduced without compromising the on-target editing simply by inserting the editing enzymes into the middle of nCas9 at tolerant sites identified using a transposon-based genetic screen. Furthermore, employing this Cas-embedding strategy, we have created a highly specific editor capable of efficient C > T editing at methylated and GC-rich sequences.


Assuntos
Proteínas Associadas a CRISPR/metabolismo , DNA/genética , Edição de Genes , Desaminases APOBEC/metabolismo , Resistência a Ampicilina/genética , Sequência de Bases , Códon de Terminação/genética , Citosina/metabolismo , Elementos de DNA Transponíveis/genética , Testes Genéticos , Células HEK293 , Humanos , Mutagênese Insercional/genética
7.
Artigo em Inglês | MEDLINE | ID: mdl-33001804

RESUMO

Accurate camera pose estimation is essential and challenging for real world dynamic 3D reconstruction and augmented reality applications. In this paper, we present a novel RGB-D SLAM approach for accurate camera pose tracking in dynamic environments. Previous methods detect dynamic components only across a short time-span of consecutive frames. Instead, we provide a more accurate dynamic 3D landmark detection method, followed by the use of long-term consistency via conditional random fields, which leverages long-term observations from multiple frames. Specifically, we first introduce an efficient initial camera pose estimation method based on distinguishing dynamic from static points using graph-cut RANSAC. These static/dynamic labels are used as priors for the unary potential in the conditional random fields, which further improves the accuracy of dynamic 3D landmark detection. Evaluation using the TUM \zjc{and Bonn RGB-D dynamic datasets shows that our approach significantly outperforms state-of-the-art methods, providing much more accurate camera trajectory estimation in a variety of highly dynamic environments. We also show that dynamic 3D reconstruction can benefit from the camera poses estimated by our RGB-D SLAM approach.

8.
EMBO J ; 39(22): e104748, 2020 11 16.
Artigo em Inglês | MEDLINE | ID: mdl-33058207

RESUMO

Programmable A > I RNA editing is a valuable tool for basic research and medicine. A variety of editors have been created, but a genetically encoded editor that is both precise and efficient has not been described to date. The trade-off between precision and efficiency is exemplified in the state of the art editor REPAIR, which comprises the ADAR2 deaminase domain fused to dCas13b. REPAIR is highly efficient, but also causes significant off-target effects. Mutations that weaken the deaminase domain can minimize the undesirable effects, but this comes at the expense of on-target editing efficiency. We have now overcome this dilemma by using a multipronged approach: We have chosen an alternative Cas protein (CasRx), inserted the deaminase domain into the middle of CasRx, and redirected the editor to the nucleus. The new editor created, dubbed REPAIRx, is precise yet highly efficient, outperforming various previous versions on both mRNA and nuclear RNA targets. Thus, REPAIRx markedly expands the RNA editing toolkit and illustrates a novel strategy for base editor optimization.


Assuntos
Edição de Genes/métodos , Edição de RNA , RNA/metabolismo , Adenosina Desaminase/genética , Células HEK293 , Humanos , Mutação , Proteínas de Ligação a RNA/genética , Transcriptoma
9.
J Org Chem ; 85(15): 10271-10282, 2020 08 07.
Artigo em Inglês | MEDLINE | ID: mdl-32664730

RESUMO

Herein, we describe a method for the synthesis of aryl-(het)aryl ketones by Rh(III)-catalyzed direct coupling between quinoline-8-carbaldehydes and (het)arylboronic acids. The method has a broad substrate scope, a high functional group tolerance, and uses commercially available starting materials. Scale-up of the reaction and subsequent synthesis of tubulin polymerization inhibitor demonstrated its utilities. A plausible mechanism was proposed on the basis of the fact that a stable cycloacylrhodium intermediate complex could be used as catalyst, and the complex reacted stoichiometrically with (het)arylboronic acids.


Assuntos
Quinolinas , Ródio , Ácidos Borônicos , Catálise , Cetonas
10.
Sci Adv ; 6(21): eaaz5216, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32671210

RESUMO

Goat domestication was critical for agriculture and civilization, but its underlying genetic changes and selection regimes remain unclear. Here, we analyze the genomes of worldwide domestic goats, wild caprid species, and historical remains, providing evidence of an ancient introgression event from a West Caucasian tur-like species to the ancestor of domestic goats. One introgressed locus with a strong signature of selection harbors the MUC6 gene, which encodes a gastrointestinally secreted mucin. Experiments revealed that the nearly fixed introgressed haplotype confers enhanced immune resistance to gastrointestinal pathogens. Another locus with a strong signal of selection may be related to behavior. The selected alleles at these two loci emerged in domestic goats at least 7200 and 8100 years ago, respectively, and increased to high frequencies concurrent with the expansion of the ubiquitous modern mitochondrial haplogroup A. Tracking these archaeologically cryptic evolutionary transformations provides new insights into the mechanisms of animal domestication.

11.
Cell Prolif ; 53(5): e12820, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32350961

RESUMO

OBJECTIVES: Recently developed CRISPR-dependent cytosine base editor (CBE), converting four codons (CAA, CAG, CGA and TGG) into stop codons without DNA double-strand breaks (DSB), serves as an efficient gene disruption strategy besides uncontrollable CRISPR-mediated frameshift. However, the detailed difference of gene knockout between the two systems has not been clarified. MATERIALS AND METHODS: Here, we selected some sgRNAs with different position background, then HEK293T cells were transfected with CBE/Cas9 plasmids together with sgRNAs. GFP-positive cells were harvested by fluorescence-activated cell sorting (FACS) 48 hours after transfection. Genomic DNA was collected for deep sequencing to analyse editing efficiency and genotype. RNA and protein were extracted to analyse gene mRNA level using qPCR analysis and Western blot. RESULTS: Here, we compared the gene disruption by CBE-mediated iSTOP with CRISPR/Cas9-mediated frameshift. We found BE-mediated gene knockout yielded fewer genotypes. BE-mediated gene editing precisely achieved silencing of two neighbouring genes, while CRISPR/Cas9 may delete the large fragment between two target sites. All of three stop codons could efficiently disrupt the target genes. It is worth notifying, Cas9-mediated gene knockout showed a more impact on neighbouring genes mRNA level than the BE editor. CONCLUSIONS: Our results reveal the differences between the two gene knockout strategies and provide useful information for choosing the appropriate gene disruption strategy.


Assuntos
Sistemas CRISPR-Cas/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Citosina/metabolismo , Mutação da Fase de Leitura/genética , Sequência de Bases , Linhagem Celular , Edição de Genes/métodos , Genótipo , Células HEK293 , Humanos , Plasmídeos/genética , RNA Mensageiro/genética , Transfecção/métodos
12.
Nat Commun ; 11(1): 2653, 2020 05 27.
Artigo em Inglês | MEDLINE | ID: mdl-32461551

RESUMO

The transcriptome of the preimplantation mouse embryo has been previously annotated by short-read sequencing, with limited coverage and accuracy. Here we utilize a low-cell number transcriptome based on the Smart-seq2 method to perform long-read sequencing. Our analysis describes additional novel transcripts and complexity of the preimplantation transcriptome, identifying 2280 potential novel transcripts from previously unannotated loci and 6289 novel splicing isoforms from previously annotated genes. Notably, these novel transcripts and isoforms with transcription start sites are enriched for an active promoter modification, H3K4me3. Moreover, we generate a more complete and precise transcriptome by combining long-read and short-read data during early embryogenesis. Based on this approach, we identify a previously undescribed isoform of Kdm4dl with a modified mRNA reading frame and a novel noncoding gene designated XLOC_004958. Depletion of Kdm4dl or XLOC_004958 led to abnormal blastocyst development. Thus, our data provide a high-resolution and more precise transcriptome during preimplantation mouse embryogenesis.


Assuntos
Blastocisto/metabolismo , Anotação de Sequência Molecular/métodos , Transcriptoma/genética , Animais , Desenvolvimento Embrionário/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Histonas/metabolismo , Histona Desmetilases com o Domínio Jumonji/genética , Camundongos , Isoformas de Proteínas/genética , Splicing de RNA/genética , RNA Longo não Codificante/genética , RNA Mensageiro/genética
13.
Cell Death Dis ; 11(2): 85, 2020 02 03.
Artigo em Inglês | MEDLINE | ID: mdl-32015323

RESUMO

Autism spectrum disorder (ASD) is a neurodevelopmental disease with a strong heritability, but recent evidence suggests that epigenetic dysregulation may also contribute to the pathogenesis of ASD. Especially, increased methylation at the MECP2 promoter and decreased MECP2 expression were observed in the brains of ASD patients. However, the causative relationship of MECP2 promoter methylation and ASD has not been established. In this study, we achieved locus-specific methylation at the transcription start site (TSS) of Mecp2 in Neuro-2a cells and in mice, using nuclease-deactivated Cas9 (dCas9) fused with DNA methyltransferase catalytic domains, together with five locus-targeting sgRNAs. This locus-specific epigenetic modification led to a reduced Mecp2 expression and a series of behavioral alterations in mice, including reduced social interaction, increased grooming, enhanced anxiety/depression, and poor performance in memory tasks. We further found that specifically increasing the Mecp2 promoter methylation in the hippocampus was sufficient to induce most of the behavioral changes. Our finding therefore demonstrated for the first time the casual relationship between locus-specific DNA methylation and diseases symptoms in vivo, warranting potential therapeutic application of epigenetic editing.


Assuntos
Transtorno do Espectro Autista/genética , Transtorno do Espectro Autista/fisiopatologia , Metilação de DNA , Proteína 2 de Ligação a Metil-CpG/genética , Regiões Promotoras Genéticas , Animais , Transtorno do Espectro Autista/metabolismo , Células Cultivadas , Regulação para Baixo , Epigênese Genética , Hipocampo/metabolismo , Hipocampo/fisiopatologia , Masculino , Camundongos , Fenótipo , Comportamento Social
14.
Mol Ther ; 28(2): 431-440, 2020 02 05.
Artigo em Inglês | MEDLINE | ID: mdl-31843453

RESUMO

Traditional CRISPR/Cas9-based gene knockouts are generated by introducing DNA double-strand breaks (DSBs), but this may cause excessive DNA damage or cell death. CRISPR-based cytosine base editors (CBEs) and adenine base editors (ABEs) can facilitate C-to-T or A-to-G exchanges, respectively, without DSBs. CBEs have been employed in a gene knockout strategy: CRISPR-STOP or i-STOP changes single nucleotides to induce in-frame stop codons. However, this strategy is not applicable for some genes, and the unwanted mutations in CBE systems have recently been reported to be surprisingly significant. As a variant, the ABE systems mediate precise editing and have only rare unwanted mutations. In this study, we implemented a new strategy to induce gene silencing (i-Silence) with an ABE-mediated start codon mutation from ATG to GTG or ACG. Using both in vitro and in vivo model systems, we showed that the i-Silence approach is efficient and precise for producing a gene knockout. In addition, the i-Silence strategy can be employed to analyze ~17,804 human genes and can be used to mimic 147 kinds of pathogenic diseases caused by start codon mutations. Altogether, compared to other methods, the ABE-based i-Silence method is a safer gene knockout strategy, and it has promising application potential.


Assuntos
Adenina/metabolismo , Códon de Iniciação , Edição de Genes , Inativação Gênica , Mutação , Sistemas CRISPR-Cas , Expressão Gênica , Técnicas de Silenciamento de Genes , Genes Reporter , Células HEK293 , Sequenciamento de Nucleotídeos em Larga Escala , Humanos
15.
FEBS Lett ; 594(8): 1319-1328, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-31837228

RESUMO

Base editors (BEs) are widely used in precise gene editing due to their simplicity and versatility. However, their efficiencies are hindered by various obstacles. Considering the chromatin microenvironment as a possible obstacle, here, we demonstrate a further development of the proxy-clustered regularly interspaced short palindromic repeats strategy, termed Proxy-BE, to increase gene editing efficiency. Specifically, a nuclease-dead Cas9 (dCas9) was bound to the sequence about 20-30 base pair away from the target site, potentially improving access to the DNA and, thus, providing a better editing microenvironment for base editors. Our findings confirm that nuclease-dead Streptococcus pyogenes Cas9 can assist the base editors SaKKH-BE3 and dCpf1-BE to double their canonical base editing efficiency. This work provides a new approach to enhance base editing, extending its scope for biological research and gene therapy.


Assuntos
Proteína 9 Associada à CRISPR/metabolismo , Edição de Genes/métodos , Proteína 9 Associada à CRISPR/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Células HEK293 , Humanos
16.
Pest Manag Sci ; 76(10): 3369-3376, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31756256

RESUMO

BACKGROUND: Food is an important strategic material related to national economy and people's livelihood. Plant diseases seriously affect crop yield and quality. Marine natural products are an important source for novel drugs discovery. In this work, meridianin alkaloids were selected as the parent structure. A series of meridianin alkaloid analogues were rationally designed, synthesized and evaluated for their antiviral activities and fungicidal activities. RESULT: These compounds were found to have good antiviral and fungicidal activities for the first time. The structure-activity relationship (SAR) research revealed that introducing bromine atom at the 5-position of indole ring is beneficial to antiviral activity, but introducing methoxy group is not conducive. Introducing bromine atom at the 6-position of indole ring or nitrogen atom at the 7-position of the indole ring resulted in lower antiviral activity. Most of the meridianin derivatives showed higher anti-TMV activities at 500 µg mL-1 than Ribavirin, especially for compounds 6c, 8a and 10a. All of the compounds also displayed broad spectrum fungicidal activities against 14 kinds of phytopathogenic fungi at 50 µg mL-1 . CONCLUSION: Compound 6c with relatively simple structure and excellent antiviral activity, which is similar to that of Ningnanmycin, emerged as novel anti-TMV lead compound. Compound 5d with broad spectrum and high effect fungicidal activity emerged as a new fungicidal lead compound. Current research lays a solid foundation for the application of meridianin alkaloids in crop protection. © 2019 Society of Chemical Industry.


Assuntos
Antivirais/farmacologia , Alcaloides , Produtos Biológicos , Desinfetantes , Desenho de Fármacos , Fungos , Estrutura Molecular , Relação Estrutura-Atividade , Vírus do Mosaico do Tabaco
17.
Mol Ther Nucleic Acids ; 17: 289-296, 2019 Sep 06.
Artigo em Inglês | MEDLINE | ID: mdl-31279230

RESUMO

Base editing systems show their power in modeling and correcting the pathogenic mutations of genetic diseases. Previous studies have already demonstrated the editing efficiency of BE3-mediated C-to-T conversion in human embryos. However, the precision and efficiency of a recently developed adenine base editor (ABE), which converts A-to-G editing in human embryos, remain to be addressed. Here we selected reported pathogenic mutations to characterize the ABE in human tripronuclear embryos. We found effective A-to-G editing occurred at the desirable sites using the ABE system. Furthermore, ABE-mediated A-to-G editing in the single blastomere of the edited embryos exhibited high product purity. By deep sequencing and whole-genome sequencing, A or T mutations didn't increase significantly, and no off-target or insertion or deletion (indel) mutations were detected in these edited embryos, indicating the ABE-mediated base editing in human embryos is precise and controllable. For some sites, since a different editing pattern was obtained from the cells and the embryos targeted with the same single guide RNA (sgRNA), it suggests that ABE-mediated editing might have different specificity in vivo. Taken together, we efficiently generated pathogenic A-to-G mutations in human tripronuclear embryos via ABE-mediated base editing.

18.
Org Lett ; 21(14): 5728-5732, 2019 Jul 19.
Artigo em Inglês | MEDLINE | ID: mdl-31251074

RESUMO

Herein we report a protocol for visible-light-induced copper-catalyzed decarboxylative coupling reactions between N-heteroarenes and redox-active esters. Various N-hydroxyphthalimide esters reacted with isoquinoline, quinoline, pyridine, pyrimidine, quinazoline, phthalazine, phenanthridine, and pyridazine to give the corresponding products in modest to excellent yields. The reactions proceed under mild conditions and have a broad scope and high functional group tolerance. Mechanistic studies revealed that the catalytic behavior of CuI photocatalyst generated in situ was consistent with that of preformed [Cu(dmp)(xantphos)]BF4.

19.
iScience ; 15: 640-648, 2019 May 31.
Artigo em Inglês | MEDLINE | ID: mdl-31130518

RESUMO

RNA splicing is related to many human diseases; however, lack of efficient genetic approaches to modulate splicing has prevented us from dissecting their functions in human diseases. Recently developed base editors (BEs) offer a new strategy to modulate RNA splicing by converting conservative splice sites, but it is limited by the editing precision and scope. To overcome the limitations of currently available BE-based tools, we combined SpCas9-NG with ABEmax to generate a new BE, ABEmax-NG. We demonstrated that ABEmax-NG performed precise A⋅T to G⋅C conversion with an expanded scope, thus covering many more splicing sites. Taking advantage of this tool, we precisely achieved A⋅T to G⋅C conversion exactly at the splice sites. We further modeled pathogenic RNA splicing in vitro and in vivo. Taken together, we successfully generated a versatile tool suitable for precise and broad editing at the splice sites.

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