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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.
J Genet Genomics ; 48(5): 347-360, 2021 05 20.
Artigo em Inglês | MEDLINE | ID: mdl-34144928

RESUMO

Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (CRISPR/Cas9) genome editing technology has dramatically influenced swine research by enabling the production of high-quality disease-resistant pig breeds, thus improving yields. In addition, CRISPR/Cas9 has been used extensively in pigs as one of the tools in biomedical research. In this review, we present the advancements of the CRISPR/Cas9 system in swine research, such as animal breeding, vaccine development, xenotransplantation, and disease modeling. We also highlight the current challenges and some potential applications of the CRISPR/Cas9 technologies.

3.
Mol Ther ; 2021 Jun 24.
Artigo em Inglês | MEDLINE | ID: mdl-34174443

RESUMO

Myosin VI(MYO6) is an unconventional myosin that is vital for auditory and vestibular function. Pathogenic variants in the human MYO6 gene cause autosomal-dominant or -recessive forms of hearing loss. Effective treatments for Myo6 mutation causing hearing loss are limited. We studied whether adeno-associated virus (AAV)-PHP.eB vector-mediated in vivo delivery of Staphylococcus aureus Cas9 (SaCas9-KKH)-single-guide RNA (sgRNA) complexes could ameliorate hearing loss in a Myo6WT/C442Y mouse model that recapitulated the phenotypes of human patients. The in vivo editing efficiency of the AAV-SaCas9-KKH-Myo6-g2 system on Myo6C442Y is 4.05% on average in Myo6WT/C442Y mice, which was ∼17-fold greater than editing efficiency of Myo6WT alleles. Rescue of auditory function was observed up to 5 months post AAV-SaCas9-KKH-Myo6-g2 injection in Myo6WT/C442Y mice. Meanwhile, shorter latencies of auditory brainstem response (ABR) wave I, lower distortion product otoacoustic emission (DPOAE) thresholds, increased cell survival rates, more regular hair bundle morphology, and recovery of inward calcium levels were also observed in the AAV-SaCas9-KKH-Myo6-g2-treated ears compared to untreated ears. These findings provide further reference for in vivo genome editing as a therapeutic treatment for various semi-dominant forms of hearing loss and other semi-dominant diseases.

6.
EMBO J ; 39(22): e104741, 2020 11 16.
Artigo em Inglês | MEDLINE | ID: mdl-33058229

RESUMO

Programmable RNA cytidine deamination has recently been achieved using a bifunctional editor (RESCUE-S) capable of deaminating both adenine and cysteine. Here, we report the development of "CURE", the first cytidine-specific C-to-U RNA Editor. CURE comprises the cytidine deaminase enzyme APOBEC3A fused to dCas13 and acts in conjunction with unconventional guide RNAs (gRNAs) designed to induce loops at the target sites. Importantly, CURE does not deaminate adenosine, enabling the high-specificity versions of CURE to create fewer missense mutations than RESCUE-S at the off-targets transcriptome-wide. The two editing approaches exhibit overlapping editing motif preferences, with CURE and RESCUE-S being uniquely able to edit UCC and AC motifs, respectively, while they outperform each other at different subsets of the UC targets. Finally, a nuclear-localized version of CURE, but not that of RESCUE-S, can efficiently edit nuclear RNAs. Thus, CURE and RESCUE are distinct in design and complementary in utility.


Assuntos
Citidina Desaminase/genética , Proteínas/genética , Edição de RNA , Núcleo Celular/metabolismo , Células HEK293 , Humanos , RNA/química , RNA/metabolismo , RNA Guia , Transcriptoma
7.
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.

8.
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
9.
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
12.
Biol Reprod ; 102(4): 817-827, 2020 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-31916576

RESUMO

Genome stability is critical for the normal development of preimplantation embryos, as DNA damages may result in mutation and even embryo lethality. Anti-silencing factor 1A (ASF1A) is a histone chaperone and enriched in the MII oocytes as a maternal factor, which may be associated with the maintenance of genome stability. Thus, this study was undertaken to explore the role of ASF1A in maintaining the genome stability of early mouse embryos. The ASF1A expressed in the preimplantation embryos and displayed a dynamic pattern throughout the early embryonic development. Inhibition of ASF1A expression decreased embryonic development and increased DNA damages. Overexpression of ASF1A improved the developmental potential and decreased DNA damages. When 293T cells that had been integrated with RGS-NHEJ were co-transfected with plasmids of pcDNA3.1-ASF1A, gRNA-NHEJ, and hCas9, less cells expressed eGFP, indicating that non-homologous end joining was reduced by ASF1A. When 293T cells were co-transfected with plasmids of HR-donor, gRNA-HR, hCas9, and pcDNA3.1-ASF1A, more cells expressed eGFP, indicating that homologous recombination (HR) was enhanced by ASF1A. These results indicate that ASF1A may be associated with the genome stability maintenance of early mouse embryos and this action may be mediated by promoting DNA damage repair through HR pathway.

13.
Sci Rep ; 9(1): 14315, 2019 10 04.
Artigo em Inglês | MEDLINE | ID: mdl-31586114

RESUMO

Although numerous attempts have been made to alter the sex ratio of the progeny of mammals, the limitations of current technologies have prevented their widespread use in farm animals. The presence or absence of a Y chromosome determines whether a mammalian embryo develops as a male or female, and non-invasive genetic reporters such as fluorescence protein markers have been intensively applied in a variety of fields of research. To develop a non-invasive and instantaneous method for advance determination of the sex of embryos, we developed a Y chromosome-linked eGFP mouse line that stably expresses green fluorescent protein under the control of the CAG promoter. The development of the CRISPR/Cas9 system has made it easy to deliver an exogenous gene to a specific locus of a genome, and linking a tracer to the Y chromosome has simplified the process of predicting the sex of embryos collected by mating a Y-Chr-eGFP transgenic male with a wild-type female. XY embryos appeared green, under a fluorescence microscope, and XX embryos did not. Y chromosome-linked genes were amplified by nested PCR to further confirm the accuracy of this method, and the simultaneous transplantation of green and non-green embryos into foster mothers indicated that 100% accuracy was achieved by this method. Thus, the Y-Chr-eGFP mouse line provides an expeditious and accurate approach for sexing pre-implantation embryos and can be efficiently used for the pre-selection of sex.


Assuntos
Sistemas CRISPR-Cas , Camundongos Transgênicos/embriologia , Análise para Determinação do Sexo , Cromossomo Y , Animais , Embrião de Mamíferos , Desenvolvimento Embrionário , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos/genética
14.
Development ; 146(13)2019 07 10.
Artigo em Inglês | MEDLINE | ID: mdl-31189663

RESUMO

Epigenetic regulation, including histone-to-protamine exchanges, controls spermiogenesis. However, the underlying mechanisms of this regulation are largely unknown. Here, we report that PHF7, a testis-specific PHD and RING finger domain-containing protein, is essential for histone-to-protamine exchange in mice. PHF7 is specifically expressed during spermiogenesis. PHF7 deletion results in male infertility due to aberrant histone retention and impaired protamine replacement in elongated spermatids. Mechanistically, PHF7 can simultaneously bind histone H2A and H3; its PHD domain, a histone code reader, can specifically bind H3K4me3/me2, and its RING domain, a histone writer, can ubiquitylate H2A. Thus, our study reveals that PHF7 is a novel E3 ligase that can specifically ubiquitylate H2A through binding H3K4me3/me2 prior to histone-to-protamine exchange.


Assuntos
Histonas/metabolismo , Protaminas/metabolismo , Espermatogênese/genética , Ubiquitina-Proteína Ligases/fisiologia , Ubiquitinação/genética , Animais , Células Cultivadas , Montagem e Desmontagem da Cromatina/fisiologia , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Infertilidade Masculina/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Transdução de Sinais/genética , Testículo/metabolismo , Ubiquitina-Proteína Ligases/genética
15.
Nature ; 571(7764): 275-278, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31181567

RESUMO

Recently developed DNA base editing methods enable the direct generation of desired point mutations in genomic DNA without generating any double-strand breaks1-3, but the issue of off-target edits has limited the application of these methods. Although several previous studies have evaluated off-target mutations in genomic DNA4-8, it is now clear that the deaminases that are integral to commonly used DNA base editors often bind to RNA9-13. For example, the cytosine deaminase APOBEC1-which is used in cytosine base editors (CBEs)-targets both DNA and RNA12, and the adenine deaminase TadA-which is used in adenine base editors (ABEs)-induces site-specific inosine formation on RNA9,11. However, any potential RNA mutations caused by DNA base editors have not been evaluated. Adeno-associated viruses are the most common delivery system for gene therapies that involve DNA editing; these viruses can sustain long-term gene expression in vivo, so the extent of potential RNA mutations induced by DNA base editors is of great concern14-16. Here we quantitatively evaluated RNA single nucleotide variations (SNVs) that were induced by CBEs or ABEs. Both the cytosine base editor BE3 and the adenine base editor ABE7.10 generated tens of thousands of off-target RNA SNVs. Subsequently, by engineering deaminases, we found that three CBE variants and one ABE variant showed a reduction in off-target RNA SNVs to the baseline while maintaining efficient DNA on-target activity. This study reveals a previously overlooked aspect of off-target effects in DNA editing and also demonstrates that such effects can be eliminated by engineering deaminases.


Assuntos
DNA/genética , Edição de Genes/métodos , Mutagênese , Mutação , Nucleosídeo Desaminases/genética , Engenharia de Proteínas , RNA/genética , Adenina/metabolismo , Aminoidrolases/genética , Aminoidrolases/metabolismo , Citosina/metabolismo , Citosina Desaminase/genética , Citosina Desaminase/metabolismo , Células HEK293 , Humanos , Nucleosídeo Desaminases/metabolismo , Especificidade por Substrato , Transfecção
16.
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
17.
J Genet Genomics ; 46(11): 513-521, 2019 11 20.
Artigo em Inglês | MEDLINE | ID: mdl-31911131

RESUMO

CRISPR-mediated genome editing is a revolutionary technology for genome manipulation that uses the CRISPR-Cas systems and base editors. Currently, poor efficiency and off-target problems have impeded the application of CRISPR systems. The on-target efficiency has been improved in several advanced versions of CRISPR systems, whereas the off-target detection still remains a key challenge. Here, we outline the different versions of CRISPR systems and off-target detection strategies, discuss the merits and limitations of off-target detection methods, and provide potential implications for further gene editing research.


Assuntos
Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Edição de Genes/métodos , Proteína 9 Associada à CRISPR/genética , Imunoprecipitação da Cromatina , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , RNA Guia/genética , Sequenciamento Completo do Genoma/métodos
18.
Genome Biol ; 18(1): 224, 2017 11 24.
Artigo em Inglês | MEDLINE | ID: mdl-29178945

RESUMO

BACKGROUND: The CRISPR/Cas9 system has become an efficient gene editing method for generating cells carrying precise gene mutations, including the rearrangement and deletion of chromosomal segments. However, whether an entire chromosome could be eliminated by this technology is still unknown. RESULTS: Here we demonstrate the use of the CRISPR/Cas9 system to eliminate targeted chromosomes. Using either multiple cleavages induced by a single-guide RNA (sgRNA) that targets multiple chromosome-specific sites or a cocktail of multiple sgRNAs, each targeting one specific site, we found that a sex chromosome could be selectively eliminated in cultured cells, embryos, and tissues in vivo. Furthermore, this approach was able to produce a targeted autosome loss in aneuploid mouse embryonic stem cells with an extra human chromosome and human induced pluripotent stem cells with trisomy 21, as well as cancer cells. CONCLUSIONS: CRISPR/Cas9-mediated targeted chromosome elimination offers a new approach to develop animal models with chromosome deletions, and a potential therapeutic strategy for human aneuploidy diseases involving additional chromosomes.


Assuntos
Sistemas CRISPR-Cas , Deleção Cromossômica , Marcação de Genes , Animais , Modelos Animais de Doenças , Células-Tronco Embrionárias , Feminino , Edição de Genes , Hibridização in Situ Fluorescente , Cariótipo , Masculino , Camundongos , Microinjeções , Fenótipo , RNA Guia , Síndrome de Turner/genética , Cromossomo Y
19.
Cell Res ; 27(7): 933-945, 2017 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-28585534

RESUMO

The CRISPR/Cas9 system is an efficient gene-editing method, but the majority of gene-edited animals showed mosaicism, with editing occurring only in a portion of cells. Here we show that single gene or multiple genes can be completely knocked out in mouse and monkey embryos by zygotic injection of Cas9 mRNA and multiple adjacent single-guide RNAs (spaced 10-200 bp apart) that target only a single key exon of each gene. Phenotypic analysis of F0 mice following targeted deletion of eight genes on the Y chromosome individually demonstrated the robustness of this approach in generating knockout mice. Importantly, this approach delivers complete gene knockout at high efficiencies (100% on Arntl and 91% on Prrt2) in monkey embryos. Finally, we could generate a complete Prrt2 knockout monkey in a single step, demonstrating the usefulness of this approach in rapidly establishing gene-edited monkey models.


Assuntos
Sistemas CRISPR-Cas/genética , Edição de Genes/métodos , Técnicas de Inativação de Genes , Haplorrinos/genética , RNA Guia/genética , Fatores de Transcrição ARNTL/genética , Animais , Proteínas de Bactérias , Proteína 9 Associada à CRISPR , Embrião de Mamíferos/citologia , Endonucleases , Éxons/genética , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mosaicismo/embriologia , Recuperação de Oócitos , Fenótipo , RNA Mensageiro/genética , Sequenciamento Completo do Genoma , Cromossomo Y , Zigoto/citologia
20.
Cell Res ; 27(6): 815-829, 2017 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-28429771

RESUMO

Studying the early function of essential genes is an important and challenging problem in developmental biology. Here, we established a method for rapidly inducing CRISPR-Cas9-mediated mutations in one blastomere of two-cell stage embryos, termed 2-cell embryo-CRISPR-Cas9 injection (2CC), to study the in vivo function of essential (or unknown) genes in founder chimeric mice. By injecting both Cre mRNA and CRISPR-Cas9 targeting the gene of interest into fluorescent reporter mice, the 2CC method can trace both wild-type and mutant cells at different developmental stages, offering internal control for phenotypic analyses of mutant cells. Using this method, we identified novel functions of the essential gene Tet3 in regulating excitatory and inhibitory synaptic transmission in the developing mouse cerebral cortex. By generating chimeric mutant mice, the 2CC method allows for the rapid screening of gene function in multiple tissues and cell types in founder chimeric mice, significantly expanding the current armamentarium of genetic tools.


Assuntos
Blastômeros/metabolismo , Sistemas CRISPR-Cas/fisiologia , Edição de Genes/métodos , Animais , Sistemas CRISPR-Cas/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Dioxigenases , Embrião de Mamíferos/metabolismo , Engenharia Genética/métodos , Masculino , Camundongos , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo
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