RESUMO
The CRISPR-Cas9 system offers targeted genome manipulation with simplicity. Combining the CRISPR-Cas9 with optogenetics technology, we have engineered photoactivatable Cas9 to precisely control the genome sequence in a spatiotemporal manner. Here we provide a detailed protocol for optogenetic genome editing experiments using photoactivatable Cas9, including that for the generation of guide RNA vectors, light-mediated Cas9 activation, and quantification of genome editing efficiency in mammalian cells.
Assuntos
Proteína 9 Associada à CRISPR/efeitos da radiação , Sistemas CRISPR-Cas/efeitos da radiação , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Edição de Genes , Regulação da Expressão Gênica/efeitos da radiação , Luz , Optogenética , Proteína 9 Associada à CRISPR/genética , Proteína 9 Associada à CRISPR/metabolismo , Técnicas de Cultura de Células , Reparo do DNA por Junção de Extremidades , Células HEK293 , Humanos , Mutação INDEL , Mutação Puntual , RNA Guia de Cinetoplastídeos/genética , RNA Guia de Cinetoplastídeos/metabolismoRESUMO
Following introduction of CRISPR-Cas9 components into a cell, genome editing occurs unabated until degradation of its component nucleic acids and proteins by cellular processes. This uncontrolled reaction can lead to unintended consequences including off-target editing and chromosomal translocations. To address this, we develop a method for light-induced degradation of sgRNA termed CRISPRoff. Here we show that light-induced inactivation of ribonucleoprotein attenuates genome editing within cells and allows for titratable levels of editing efficiency and spatial patterning via selective illumination.
Assuntos
Sistemas CRISPR-Cas/genética , Edição de Genes/métodos , Luz , Estabilidade de RNA/efeitos da radiação , RNA Guia de Cinetoplastídeos/metabolismo , Sistemas CRISPR-Cas/efeitos da radiação , Linhagem Celular Tumoral , Quebras de DNA de Cadeia Dupla , Estudos de Viabilidade , Células HEK293 , Humanos , RNA Guia de Cinetoplastídeos/efeitos da radiação , Ribonucleoproteínas/metabolismo , Translocação GenéticaRESUMO
As one of the most favorable stimuli, photoactivation provides an advantageous way to manipulate biological objects. In the current study, we have successfully demonstrated the use of light activation guide RNA (gRNA) strategy for controlling CRISPR systems. By conjugating photolabile protecting groups, the CRISPR functions became minimal, but exposure of acylated gRNAs to 365 nm light triggers the removal of masking groups, leading to the rescue of CRISPR functions. Furthermore, our strategy has been successfully used to control gene editing in human cells. This proof-of-concept study therefore demonstrates the promising potential of our strategy to versatile applications in chemical biology.
Assuntos
Sistemas CRISPR-Cas/efeitos da radiação , Edição de Genes/métodos , Luz , RNA Guia de Cinetoplastídeos/genética , Acetilação/efeitos da radiação , Linhagem Celular , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/efeitos da radiação , Humanos , Clivagem do RNA/efeitos da radiação , RNA Guia de Cinetoplastídeos/químicaRESUMO
We herein report an optogenetically activatable CRISPR-Cas9 nanosystem for programmable genome editing in the second near-infrared (NIR-II) optical window. The nanosystem, termed nanoCRISPR, is composed of a cationic polymer-coated Au nanorod (APC) and Cas9 plasmid driven by a heat-inducible promoter. The APC not only serves as a carrier for intracellular plasmid delivery but also can harvest external NIR-II photonic energy and convert it into local heat to induce the gene expression of the Cas9 endonuclease. Due to high transfection activity, the APC shows strong ability to induce a significant level of disruption in different genomic loci upon optogenetic activation. Moreover, the precise control of genome-editing activity can be simply programmed by finely tuning exposure time and irradiation time in vitro and in vivo and also enables editing at multiple time points, thus proving the sensitivity and inducibility of such an editing modality. The NIR-II optical feature of nanoCRISPR enables therapeutic genome editing at deep tissue, by which treatment of deep tumor and rescue of fulminant hepatic failure are demonstrated as proof-of-concept therapeutic examples. Importantly, this modality of optogenetic genome editing can significantly minimize the off-target effect of CRISPR-Cas9 in most potential off-target sites. The optogenetically activatable CRISPR-Cas9 nanosystem we have developed offers a useful tool to expand the current applications of CRISPR-Cas9, and also defines a programmable genome-editing strategy toward high precision and spatial specificity.
Assuntos
Sistemas CRISPR-Cas , Edição de Genes/métodos , Nanotubos/química , Optogenética , Proteína 9 Associada à CRISPR/genética , Sistemas CRISPR-Cas/genética , Sistemas CRISPR-Cas/efeitos da radiação , Ouro/química , Células HEK293 , Humanos , Raios Infravermelhos , Plasmídeos/genética , Regiões Promotoras GenéticasRESUMO
As an RNA-guided nuclease, CRISPR-Cas9 offers facile and promising solutions to mediate genome modification with respect to versatility and high precision. However, spatiotemporal manipulation of CRISPR-Cas9 delivery remains a daunting challenge for robust effectuation of gene editing both in vitro and in vivo. Here, we designed a near-infrared (NIR) light-responsive nanocarrier of CRISPR-Cas9 for cancer therapeutics based on upconversion nanoparticles (UCNPs). The UCNPs served as "nanotransducers" that can convert NIR light (980 nm) into local ultraviolet light for the cleavage of photosensitive molecules, thereby resulting in on-demand release of CRISPR-Cas9. In addition, by preparing a single guide RNA targeting a tumor gene (polo-like kinase-1), our strategies have successfully inhibited the proliferation of tumor cell via NIR light-activated gene editing both in vitro and in vivo. Overall, this exogenously controlled method presents enormous potential for targeted gene editing in deep tissues and treatment of a myriad of diseases.
Assuntos
Sistemas CRISPR-Cas/genética , Carcinoma de Células Escamosas/terapia , Portadores de Fármacos/química , Edição de Genes/métodos , Raios Infravermelhos , Neoplasias Bucais/terapia , Nanopartículas/administração & dosagem , Animais , Apoptose , Sistemas CRISPR-Cas/efeitos da radiação , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patologia , Proliferação de Células , Feminino , Técnicas de Transferência de Genes , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Neoplasias Bucais/genética , Neoplasias Bucais/patologia , Nanopartículas/química , RNA Guia de Cinetoplastídeos/genética , RNA Guia de Cinetoplastídeos/efeitos da radiação , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Gene therapy is expected to be utilized for the treatment of various diseases. However, the spatiotemporal resolution of current gene therapy technology is not high enough. In this study, we generated a new technology for spatiotemporally controllable gene therapy. We introduced optogenetic and CRISPR/Cas9 techniques into a recombinant adenovirus (Ad) vector, which is widely used in clinical trials and exhibits high gene transfer efficiency, to generate an illumination-dependent spatiotemporally controllable gene regulation system (designated the Opt/Cas-Ad system). We generated an Opt/Cas-Ad system that could regulate a potential tumor suppressor gene, and we examined the effectiveness of this system in cancer treatment using a xenograft tumor model. With the Opt/Cas-Ad system, highly selective tumor treatment could be performed by illuminating the tumor. In addition, Opt/Cas-Ad system-mediated tumor treatment could be stopped simply by turning off the light. We believe that our Opt/Cas-Ad system can enhance both the safety and effectiveness of gene therapy.
Assuntos
Adenoviridae/genética , Proteínas Associadas a CRISPR/genética , Proteínas Associadas a CRISPR/efeitos da radiação , Sistemas CRISPR-Cas/genética , Sistemas CRISPR-Cas/efeitos da radiação , Terapia Genética/métodos , Proteínas Adaptadoras de Transdução de Sinal , Animais , Linhagem Celular Tumoral , Quimiocinas , Endonucleases/genética , Endonucleases/efeitos da radiação , Feminino , Regulação da Expressão Gênica , Técnicas de Transferência de Genes , Genes Supressores de Tumor/efeitos da radiação , Vetores Genéticos , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/genética , Luz , Camundongos Endogâmicos BALB C , RNA Guia de Cinetoplastídeos/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Optical control of CRISPR-Cas9-derived proteins would be useful for restricting gene editing or transcriptional regulation to desired times and places. Optical control of Cas9 functions has been achieved with photouncageable unnatural amino acids or by using light-induced protein interactions to reconstitute Cas9-mediated functions from two polypeptides. However, these methods have only been applied to one Cas9 species and have not been used for optical control of different perturbations at two genes. Here, we use photodissociable dimeric fluorescent protein domains to engineer single-chain photoswitchable Cas9 (ps-Cas9) proteins in which the DNA-binding cleft is occluded at baseline and opened upon illumination. This design successfully controlled different species and functional variants of Cas9, mediated transcriptional activation more robustly than previous optogenetic methods, and enabled light-induced transcription of one gene and editing of another in the same cells. Thus, a single-chain photoswitchable architecture provides a general method to control a variety of Cas9-mediated functions.
Assuntos
Proteína 9 Associada à CRISPR/genética , Proteínas Associadas a CRISPR/genética , Edição de Genes/métodos , Proteínas de Fluorescência Verde/genética , Proteína 9 Associada à CRISPR/química , Proteína 9 Associada à CRISPR/efeitos da radiação , Proteínas Associadas a CRISPR/química , Proteínas Associadas a CRISPR/efeitos da radiação , Sistemas CRISPR-Cas/genética , Sistemas CRISPR-Cas/efeitos da radiação , Regulação da Expressão Gênica , Proteínas de Fluorescência Verde/efeitos da radiação , Células HEK293 , Humanos , Luz , Mutação , Domínios Proteicos/genética , Engenharia de Proteínas , Streptococcus pyogenes/enzimologia , Transcrição GênicaRESUMO
We describe an engineered photoactivatable Cas9 (paCas9) that enables optogenetic control of CRISPR-Cas9 genome editing in human cells. paCas9 consists of split Cas9 fragments and photoinducible dimerization domains named Magnets. In response to blue light irradiation, paCas9 expressed in human embryonic kidney 293T cells induces targeted genome sequence modifications through both nonhomologous end joining and homology-directed repair pathways. Genome editing activity can be switched off simply by extinguishing the light. We also demonstrate activation of paCas9 in spatial patterns determined by the sites of irradiation. Optogenetic control of targeted genome editing should facilitate improved understanding of complex gene networks and could prove useful in biomedical applications.
Assuntos
Sistemas CRISPR-Cas/genética , Sistemas CRISPR-Cas/efeitos da radiação , Engenharia Genética/métodos , Optogenética/métodos , Sequência de Bases , Células HEK293 , Humanos , Modelos Genéticos , Dados de Sequência Molecular , Interferência de RNARESUMO
Optogenetic systems enable precise spatial and temporal control of cell behavior. We engineered a light-activated CRISPR-Cas9 effector (LACE) system that induces transcription of endogenous genes in the presence of blue light. This was accomplished by fusing the light-inducible heterodimerizing proteins CRY2 and CIB1 to a transactivation domain and the catalytically inactive dCas9, respectively. The versatile LACE system can be easily directed to new DNA sequences for the dynamic regulation of endogenous genes.