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1.
Cell ; 184(22): 5635-5652.e29, 2021 10 28.
Artigo em Inglês | MEDLINE | ID: mdl-34653350

RESUMO

While prime editing enables precise sequence changes in DNA, cellular determinants of prime editing remain poorly understood. Using pooled CRISPRi screens, we discovered that DNA mismatch repair (MMR) impedes prime editing and promotes undesired indel byproducts. We developed PE4 and PE5 prime editing systems in which transient expression of an engineered MMR-inhibiting protein enhances the efficiency of substitution, small insertion, and small deletion prime edits by an average 7.7-fold and 2.0-fold compared to PE2 and PE3 systems, respectively, while improving edit/indel ratios by 3.4-fold in MMR-proficient cell types. Strategic installation of silent mutations near the intended edit can enhance prime editing outcomes by evading MMR. Prime editor protein optimization resulted in a PEmax architecture that enhances editing efficacy by 2.8-fold on average in HeLa cells. These findings enrich our understanding of prime editing and establish prime editing systems that show substantial improvement across 191 edits in seven mammalian cell types.


Assuntos
Edição de Genes , Sistemas CRISPR-Cas/genética , Linhagem Celular , DNA/metabolismo , Reparo de Erro de Pareamento de DNA/genética , Feminino , Genes Dominantes , Genoma Humano , Humanos , Masculino , Modelos Biológicos , Proteína 1 Homóloga a MutL/genética , Mutação/genética , RNA/metabolismo , Reprodutibilidade dos Testes
2.
Mol Ther ; 30(1): 130-144, 2022 01 05.
Artigo em Inglês | MEDLINE | ID: mdl-34737067

RESUMO

Disruption of CCR5 or CXCR4, the main human immunodeficiency virus type 1 (HIV-1) co-receptors, has been shown to protect primary human CD4+ T cells from HIV-1 infection. Base editing can install targeted point mutations in cellular genomes, and can thus efficiently inactivate genes by introducing stop codons or eliminating start codons without double-stranded DNA break formation. Here, we applied base editors for individual and simultaneous disruption of both co-receptors in primary human CD4+ T cells. Using cytosine base editors we observed premature stop codon introduction in up to 89% of sequenced CCR5 or CXCR4 alleles. Using adenine base editors we eliminated the start codon in CCR5 in up to 95% of primary human CD4+ T cell and up to 88% of CD34+ hematopoietic stem and progenitor cell target alleles. Genome-wide specificity analysis revealed low numbers of off-target mutations that were introduced by base editing, located predominantly in intergenic or intronic regions. We show that our editing strategies prevent transduction with CCR5-tropic and CXCR4-tropic viral vectors in up to 79% and 88% of human CD4+ T cells, respectively. The engineered T cells maintained functionality and overall our results demonstrate the effectiveness of base-editing strategies for efficient and specific ablation of HIV co-receptors in clinically relevant cell types.


Assuntos
Edição de Genes , Receptores CCR5 , Receptores CXCR4 , Edição de Genes/métodos , Infecções por HIV/genética , Infecções por HIV/metabolismo , Infecções por HIV/terapia , HIV-1/fisiologia , Células-Tronco Hematopoéticas/metabolismo , Humanos , Receptores CCR5/genética , Receptores CCR5/metabolismo , Receptores CXCR4/genética , Receptores CXCR4/metabolismo , Linfócitos T/metabolismo
3.
Mol Ther ; 24(3): 570-81, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26502778

RESUMO

Present adoptive immunotherapy strategies are based on the re-targeting of autologous T-cells to recognize tumor antigens. As T-cell properties may vary significantly between patients, this approach can result in significant variability in cell potency that may affect therapeutic outcome. More consistent results could be achieved by generating allogeneic cells from healthy donors. An impediment to such an approach is the endogenous T-cell receptors present on T-cells, which have the potential to direct dangerous off-tumor antihost reactivity. To address these limitations, we assessed the ability of three different TCR-α-targeted nucleases to disrupt T-cell receptor expression in primary human T-cells. We optimized the conditions for the delivery of each reagent and assessed off-target cleavage. The megaTAL and CRISPR/Cas9 reagents exhibited the highest disruption efficiency combined with low levels of toxicity and off-target cleavage, and we used them for a translatable manufacturing process to produce safe cellular substrates for next-generation immunotherapies.


Assuntos
Sistemas CRISPR-Cas , Endonucleases , Edição de Genes , Receptores de Antígenos de Linfócitos T/genética , Nucleases dos Efetores Semelhantes a Ativadores de Transcrição , Sítios de Ligação , Técnicas de Cultura de Células , Linhagem Celular , Marcação de Genes , Técnicas de Transferência de Genes , Loci Gênicos , Genoma , Humanos , Imunofenotipagem , Neoplasias/genética , Neoplasias/imunologia , Neoplasias/metabolismo , Fenótipo , Ligação Proteica , Proteínas Recombinantes de Fusão , Linfócitos T/metabolismo , Transdução Genética
4.
Bioengineering (Basel) ; 8(2)2021 Feb 12.
Artigo em Inglês | MEDLINE | ID: mdl-33673107

RESUMO

Clustered regularly interspaced short palindromic repeat (CRISPR/Cas) proteins can be designed to bind specified DNA and RNA sequences and hold great promise for the accurate detection of nucleic acids for diagnostics. We integrated commercially available reagents into a CRISPR/Cas9-based lateral flow assay that can detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) sequences with single-base specificity. This approach requires minimal equipment and represents a simplified platform for field-based deployment. We also developed a rapid, multiplex fluorescence CRISPR/Cas9 nuclease cleavage assay capable of detecting and differentiating SARS-CoV-2, influenza A and B, and respiratory syncytial virus in a single reaction. Our findings provide proof-of-principle for CRISPR/Cas9 point-of-care diagnosis as well as a scalable fluorescent platform for identifying respiratory viral pathogens with overlapping symptomology.

5.
J Invest Dermatol ; 140(2): 338-347.e5, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31437443

RESUMO

Genome editing represents a promising strategy for the therapeutic correction of COL7A1 mutations that cause recessive dystrophic epidermolysis bullosa (RDEB). DNA cleavage followed by homology-directed repair (HDR) using an exogenous template has previously been used to correct COL7A1 mutations. HDR rates can be modest, and the double-strand DNA breaks that initiate HDR commonly result in accompanying undesired insertions and deletions (indels). To overcome these limitations, we applied an A•T→G•C adenine base editor (ABE) to correct two different COL7A1 mutations in primary fibroblasts derived from RDEB patients. ABE enabled higher COL7A1 correction efficiencies than previously reported HDR efforts. Moreover, ABE obviated the need for a repair template, and minimal indels or editing at off-target sites was detected. Base editing restored the endogenous type VII collagen expression and function in vitro. We also treated induced pluripotent stem cells (iPSCs) derived from RDEB fibroblasts with ABE. The edited iPSCs were differentiated into mesenchymal stromal cells, a cell population with therapeutic potential for RDEB. In a mouse teratoma model, the skin derived from ABE-treated iPSCs showed the proper deposition of C7 at the dermal-epidermal junction in vivo. These demonstrate that base editing provides an efficient and precise genome editing method for autologous cell engineering for RDEB.


Assuntos
Engenharia Celular/métodos , Colágeno Tipo VII/genética , Epidermólise Bolhosa Distrófica/terapia , Transplante de Células-Tronco Mesenquimais , Reparo Gênico Alvo-Dirigido , Teratoma/terapia , Animais , Diferenciação Celular , Células Cultivadas , Colágeno Tipo VII/metabolismo , Modelos Animais de Doenças , Epidermólise Bolhosa Distrófica/genética , Epidermólise Bolhosa Distrófica/patologia , Fibroblastos/patologia , Genes Recessivos/genética , Humanos , Células-Tronco Pluripotentes Induzidas/fisiologia , Células-Tronco Mesenquimais/fisiologia , Camundongos , Mutação , Cultura Primária de Células , Teratoma/genética , Teratoma/patologia , Transfecção , Transplante Autólogo/métodos
6.
Mol Ther Methods Clin Dev ; 4: 213-224, 2017 Mar 17.
Artigo em Inglês | MEDLINE | ID: mdl-28345006

RESUMO

In T cells with transgenic high-avidity T cell receptors (TCRs), endogenous and transferred TCR chains compete for surface expression and may pair inappropriately, potentially causing autoimmunity. To knock out endogenous TCR expression, we assembled 12 transcription activator-like effector nucleases (TALENs) and five guide RNAs (gRNAs) from the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas9) system. Using TALEN mRNA, TCR knockout was successful in up to 81% of T cells. Additionally, we were able to verify targeted gene addition of a GFP gene by homology-directed repair at the TALEN target site, using a donor suitable for replacement of the reporter transgene with therapeutic TCR chains. Remarkably, analysis of TALEN and CRISPR/Cas9 specificity using integrase-defective lentiviral vector capture revealed only one off-target site for one of the gRNAs and three off-target sites for both of the TALENs, indicating a high level of specificity. Collectively, our work shows highly efficient and specific nucleases for T cell engineering.

7.
Vaccine ; 28(42): 6930-41, 2010 Oct 04.
Artigo em Inglês | MEDLINE | ID: mdl-20728521

RESUMO

HIV-Tat based vaccines have been proposed as an attractive option to prevent or treat AIDS. A vaccine to induce optimal anti-Tat neutralizing antibody responses was designed by inserting this protein, or its dominant B-cell epitope, into the CyaA vector, which targets dendritic cells (DC). Tat was inserted into various sites of CyaA, including regions that do not translocate into the cytosol of the targeted DC. The presentation of the Tat CD4(+) T-cell epitope delivered by the CyaA-Tat proteins was observed with a recombinant CyaA in which the entire AC domain was replaced by the entire Tat protein (Tat-Δ373 CyaA) but was abolished with large deletions of the N-terminal region. Moreover, CyaA carrying multiple copies of the dominant Tat: 1-21 B-cell epitope were shown to induce high titers of anti-Tat antibodies, even after a single immunization, that persisted up to 10 weeks post-immunization.


Assuntos
Vacinas contra a AIDS/imunologia , Anticorpos Neutralizantes/sangue , Células Dendríticas/imunologia , Epitopos de Linfócito B/imunologia , Produtos do Gene tat do Vírus da Imunodeficiência Humana/imunologia , Vacinas contra a AIDS/genética , Toxina Adenilato Ciclase/imunologia , Animais , Apresentação de Antígeno , Células CHO , Ilhas de CpG , Cricetinae , Cricetulus , Citocinas/imunologia , Epitopos de Linfócito T/imunologia , Feminino , Anticorpos Anti-HIV/sangue , Imunidade Celular , Camundongos , Camundongos Endogâmicos BALB C , Testes de Neutralização , Poli I-C/imunologia , Proteínas Recombinantes/imunologia , Produtos do Gene tat do Vírus da Imunodeficiência Humana/genética
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