RESUMEN
The utilization of electroporation for delivering CRISPR/Cas9 system components has enabled efficient gene editing in mammalian zygotes, facilitating the development of genome-edited animals. In this study, our research focused on targeting the ACTG1 and MSTN genes in sheep, revealing a threshold phenomenon in electroporation with a voltage tolerance in sheep in vitro fertilization (IVF) zygotes. Various poring voltages near 40 V and pulse durations were examined for electroporating sheep zygotes. The study concluded that stronger electric fields required shorter pulse durations to achieve the optimal conditions for high gene mutation rates and reasonable blastocyst development. This investigation also assessed the quality of Cas9/sgRNA ribonucleoprotein complexes (Cas9 RNPs) and their influence on genome editing efficiency in sheep early embryos. It was highlighted that pre-complexation of Cas9 proteins with single-guide RNA (sgRNA) before electroporation was essential for achieving a high mutation rate. The use of suitable electroporation parameters for sheep IVF zygotes led to significantly high mutation rates and heterozygote ratios. By delivering Cas9 RNPs and single-stranded oligodeoxynucleotides (ssODNs) to zygotes through electroporation, targeting the MSTN (Myostatin) gene, a knock-in efficiency of 26% was achieved. The successful generation of MSTN-modified lambs was demonstrated by delivering Cas9 RNPs into IVF zygotes via electroporation.
Asunto(s)
Sistemas CRISPR-Cas , Electroporación , Fertilización In Vitro , Edición Génica , ARN Guía de Sistemas CRISPR-Cas , Ribonucleoproteínas , Cigoto , Animales , Edición Génica/métodos , Electroporación/métodos , Cigoto/metabolismo , Fertilización In Vitro/métodos , Ribonucleoproteínas/genética , Ribonucleoproteínas/metabolismo , ARN Guía de Sistemas CRISPR-Cas/genética , Ovinos , Proteína 9 Asociada a CRISPR/metabolismo , Proteína 9 Asociada a CRISPR/genética , Miostatina/genética , Femenino , Animales Modificados GenéticamenteRESUMEN
CRISPR/Cas9 technology is expected to offer novel genome editing-related therapies for various diseases. We previously showed that an adenovirus vector (AdV) possessing eight expression units of multiplex guide RNAs (gRNAs) was obtained with no deletion of these units. Here, we attempted to construct "all-in-one" AdVs possessing expression units of four and eight gRNAs with Cas9 nickase, although we expected obstacles to obtain complete all-in-one AdVs. The first expected obstacle was that extremely high copies of viral genomes during replication may cause severe off-target cleavages of host cells and induce homologous recombination. However, surprisingly, four units in the all-in-one AdV genome were maintained completely intact. Second, for the all-in-one AdV containing eight gRNA units, we enlarged the E3 deletion in the vector backbone and shortened the U6 promoter of the gRNA expression units to shorten the AdV genome within the adenovirus packaging limits. The final size of the all-in-one AdV genome containing eight gRNA units still slightly exceeded the reported upper limit. Nevertheless, approximately one-third of the eight units remained intact, even upon preparation for in vivo experiments. Third, the genome editing efficiency unexpectedly decreased upon enlarging the E3 deletion. Our results suggested that complete all-in-one AdVs containing four gRNA units could be obtained if the problem of the low genome editing efficiency is solved, and those containing even eight gRNA units could be obtained if the obstacle of the vector size is also removed.
Asunto(s)
Adenoviridae , Sistemas CRISPR-Cas , Edición Génica , Vectores Genéticos , ARN Guía de Sistemas CRISPR-Cas , ARN Guía de Sistemas CRISPR-Cas/genética , Vectores Genéticos/genética , Adenoviridae/genética , Edición Génica/métodos , Humanos , Células HEK293 , Genoma Viral , Proteína 9 Asociada a CRISPR/metabolismo , Proteína 9 Asociada a CRISPR/genética , Desoxirribonucleasa I/metabolismo , Desoxirribonucleasa I/genéticaRESUMEN
RNA polymerase II (Pol II) dysfunction is frequently implied in human disease. Understanding its functional mechanism is essential for designing innovative therapeutic strategies. To visualize its supra-molecular interactions with genes and nascent RNA, we generated a human cell line carrying ~335 consecutive copies of a recombinant ß-globin gene. Confocal microscopy showed that Pol II was not homogeneously concentrated around these identical gene copies. Moreover, Pol II signals partially overlapped with the genes and their nascent RNA, revealing extensive compartmentalization. Using a cell line carrying a single copy of the ß-globin gene, we also tested if the binding of catalytically dead CRISPR-associated system 9 (dCas9) to different gene regions affected Pol II transcriptional activity. We assessed Pol II localization and nascent RNA levels using chromatin immunoprecipitation and droplet digital reverse transcription PCR, respectively. Some enrichment of transcriptionally paused Pol II accumulated in the promoter region was detected in a strand-specific way of gRNA binding, and there was no decrease in nascent RNA levels. Pol II preserved its transcriptional activity in the presence of DNA-bound dCas9. Our findings contribute further insight into the complex mechanism of mRNA transcription in human cells.
Asunto(s)
ARN Polimerasa II , Transcripción Genética , Globinas beta , Humanos , ARN Polimerasa II/metabolismo , ARN Polimerasa II/genética , Globinas beta/genética , Globinas beta/metabolismo , ADN/metabolismo , ADN/genética , Regiones Promotoras Genéticas , Proteína 9 Asociada a CRISPR/metabolismo , Proteína 9 Asociada a CRISPR/genética , Sistemas CRISPR-Cas , ARN/genética , ARN/metabolismo , ARN Guía de Sistemas CRISPR-Cas/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Línea CelularRESUMEN
The thermodynamic landscape of the CRISPR/Cas9 system plays a crucial role in understanding and optimizing the performance of this revolutionary genome-editing technology. In this research, we utilized isothermal titration calorimetry and microscale thermophoresis techniques to thoroughly investigate the thermodynamic properties governing CRISPR/Cas9 interactions. Our findings revealed that the binding between sgRNA and Cas9 is primarily governed by entropy, which compensates for an unfavorable enthalpy change. Conversely, the interaction between the CRISPR RNP complex and the target DNA is characterized by a favorable enthalpy change, offsetting an unfavorable entropy change. Notably, both interactions displayed negative heat capacity changes, indicative of potential hydration, ionization, or structural rearrangements. However, we noted that the involvement of water molecules and counterions in the interactions is minimal, suggesting that structural rearrangements play a significant role in influencing the binding thermodynamics. These results offer a nuanced understanding of the energetic contributions and structural dynamics underlying CRISPR-mediated gene editing. Such insights are invaluable for optimizing the efficiency and specificity of CRISPR-based genome editing applications, ultimately advancing our ability to precisely manipulate genetic material in various organisms for research, therapeutic, and biotechnological purposes.
Asunto(s)
Sistemas CRISPR-Cas , Edición Génica , Termodinámica , Edición Génica/métodos , ADN/química , ADN/metabolismo , ADN/genética , Calorimetría , Proteína 9 Asociada a CRISPR/metabolismo , Proteína 9 Asociada a CRISPR/genética , Proteína 9 Asociada a CRISPR/química , ARN Guía de Sistemas CRISPR-Cas/química , ARN Guía de Sistemas CRISPR-Cas/metabolismo , ARN Guía de Sistemas CRISPR-Cas/genéticaRESUMEN
Vascular endothelial growth factor receptor (VEGFR)-2 is a key switch for angiogenesis, which is observed in various human diseases. In this study, a novel system for advanced prime editing (PE), termed PE6h, is developed, consisting of dual lentiviral vectors: (1) a clustered regularly interspaced palindromic repeat-associated protein 9 (H840A) nickase fused with reverse transcriptase and an enhanced PE guide RNA and (2) a dominant negative (DN) MutL homolog 1 gene with nicking guide RNA. PE6h was used to edit VEGFR2 (c.18315T>A, 50.8%) to generate a premature stop codon (TAG from AAG), resulting in the production of DN-VEGFR2 (787 aa) in human retinal microvascular endothelial cells (HRECs). DN-VEGFR2 impeded VEGF-induced phosphorylation of VEGFR2, Akt, and extracellular signal-regulated kinase-1/2 and tube formation in PE6h-edited HRECs in vitro. Overall, our results highlight the potential of PE6h to inhibit angiogenesis in vivo.
Asunto(s)
Angiogénesis , Células Endoteliales , Edición Génica , Receptor 2 de Factores de Crecimiento Endotelial Vascular , Humanos , Angiogénesis/metabolismo , Proteína 9 Asociada a CRISPR/metabolismo , Proteína 9 Asociada a CRISPR/genética , Sistemas CRISPR-Cas , Células Endoteliales/metabolismo , Edición Génica/métodos , Vectores Genéticos , Neovascularización Patológica/metabolismo , Fosforilación , Retina/metabolismo , ARN Guía de Sistemas CRISPR-Cas , Factor A de Crecimiento Endotelial Vascular/metabolismo , Factor A de Crecimiento Endotelial Vascular/genética , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismo , Receptor 2 de Factores de Crecimiento Endotelial Vascular/genéticaRESUMEN
Type I CRISPR-Cas systems are widespread and have exhibited high versatility and efficiency in genome editing and gene regulation in prokaryotes. However, due to the multi-subunit composition and large size, their application in eukaryotes has not been thoroughly investigated. Here, we demonstrate that the type I-F2 Cascade, the most compact among type I systems, with a total gene size smaller than that of SpCas9, can be developed for transcriptional activation in human cells. The efficiency of the engineered I-F2 tool can match or surpass that of dCas9. Additionally, we create a base editor using the I-F2 Cascade, which induces a considerably wide editing window (~30 nt) with a bimodal distribution. It can expand targetable sites, which is useful for disrupting functional sequences and genetic screening. This research underscores the application of compact type I systems in eukaryotes, particularly in the development of a base editor with a wide editing window.
Asunto(s)
Sistemas CRISPR-Cas , Edición Génica , Activación Transcripcional , Humanos , Edición Génica/métodos , Células HEK293 , Proteína 9 Asociada a CRISPR/metabolismo , Proteína 9 Asociada a CRISPR/genética , Ingeniería Genética/métodos , ARN Guía de Sistemas CRISPR-Cas/genéticaRESUMEN
Despite the potential of small molecules and recombinant proteins to enhance the efficiency of homology-directed repair (HDR), single-stranded DNA (ssDNA) donors, as currently designed and chemically modified, remain suboptimal for precise gene editing. Here, we screen the biased ssDNA binding sequences of DNA repair-related proteins and engineer RAD51-preferred sequences into HDR-boosting modules for ssDNA donors. Donors with these modules exhibit an augmented affinity for RAD51, thereby enhancing HDR efficiency across various genomic loci and cell types when cooperated with Cas9, nCas9, and Cas12a. By combining with an inhibitor of non-homologous end joining (NHEJ) or the HDRobust strategy, these modular ssDNA donors achieve up to 90.03% (median 74.81%) HDR efficiency. The HDR-boosting modules targeting an endogenous protein enable a chemical modification-free strategy to improve the efficacy of ssDNA donors for precise gene editing.
Asunto(s)
ADN de Cadena Simple , Edición Génica , Recombinasa Rad51 , Reparación del ADN por Recombinación , ADN de Cadena Simple/metabolismo , ADN de Cadena Simple/genética , Humanos , Edición Génica/métodos , Recombinasa Rad51/metabolismo , Recombinasa Rad51/genética , Sistemas CRISPR-Cas , Células HEK293 , Proteínas Asociadas a CRISPR/metabolismo , Proteínas Asociadas a CRISPR/genética , Proteína 9 Asociada a CRISPR/metabolismo , Proteína 9 Asociada a CRISPR/genética , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Endodesoxirribonucleasas/metabolismo , Endodesoxirribonucleasas/genética , Reparación del ADN por Unión de ExtremidadesRESUMEN
The genome-editing efficiency of the CRISPR-Cas9 system hinges on the recognition of the protospacer adjacent motif (PAM) sequence, which is essential for Cas9 binding to DNA. The commonly used Streptococcus pyogenes (SpyCas9) targets the 5'-NGG-3' PAM sequence, which does not cover all the potential genomic-editing sites. To expand the toolbox for genome editing, SpyCas9 has been engineered to recognize flexible PAM sequences and Cas9 orthologs have been used to recognize novel PAM sequences. In this study, Abyssicoccus albus Cas9 (AalCas9, 1059 aa), which is smaller than SpyCas9, was found to recognize a unique 5'-NNACR-3' PAM sequence. Modification of the guide RNA sequence improved the efficiency of AalCas9-mediated genome editing in both plant and human cells. Predicted structure-assisted introduction of a point mutation in the putative PAM recognition site shifted the sequence preference of AalCas9. These results provide insights into Cas9 diversity and novel tools for genome editing.
Asunto(s)
Proteína 9 Asociada a CRISPR , Sistemas CRISPR-Cas , Edición Génica , ARN Guía de Sistemas CRISPR-Cas , Streptococcus pyogenes , Edición Génica/métodos , Proteína 9 Asociada a CRISPR/metabolismo , Proteína 9 Asociada a CRISPR/genética , Humanos , ARN Guía de Sistemas CRISPR-Cas/genética , Streptococcus pyogenes/genética , Streptococcus pyogenes/enzimología , Motivos de Nucleótidos , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente EspaciadasRESUMEN
Many CRISPR-Cas (clustered regularly interspaced short palindromic repeats and CRISPR-associated protein) systems, which provide bacteria with adaptive immunity against phages, are transcriptionally repressed in their native hosts. How CRISPR-Cas expression is induced as needed, for example, during a bacteriophage infection, remains poorly understood. In Streptococcus pyogenes, a non-canonical guide RNA tracr-L directs Cas9 to autorepress its own promoter. Here we describe a dynamic subpopulation of cells harbouring single mutations that disrupt Cas9 binding and cause CRISPR-Cas overexpression. Cas9 actively expands this population by elevating mutation rates at the tracr-L target site. Overexpressers show higher rates of memory formation, stronger potency of old memories and a larger memory storage capacity relative to wild-type cells, which are surprisingly vulnerable to phage infection. However, in the absence of phage, CRISPR-Cas overexpression reduces fitness. We propose that CRISPR-Cas overexpressers are critical players in phage defence, enabling bacterial populations to mount rapid transcriptional responses to phage without requiring transient changes in any one cell.
Asunto(s)
Bacteriófagos , Sistemas CRISPR-Cas , Streptococcus pyogenes , Streptococcus pyogenes/genética , Streptococcus pyogenes/virología , Bacteriófagos/genética , Bacteriófagos/fisiología , Proteína 9 Asociada a CRISPR/genética , Proteína 9 Asociada a CRISPR/metabolismo , Mutación , ARN Guía de Sistemas CRISPR-Cas/genética , ARN Guía de Sistemas CRISPR-Cas/metabolismo , Regiones Promotoras Genéticas , Fagos de Streptococcus/genética , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/genética , Regulación Bacteriana de la Expresión GénicaRESUMEN
Genome editing tools, particularly the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) systems (e.g., CRISPR/Cas9), and their repurposing into epigenetic editing platforms, offer enormous potential as safe and customizable therapies for cancer. Specifically, various transcriptional abnormalities in human malignancies, such as silencing of tumor suppressors and ectopic re-expression of oncogenes, have been successfully targeted with virtually no off-target effects using CRISPR activation and repression systems. In these systems, the nuclease-deactivated Cas9 protein (dCas9) is fused to one or more domains inducing selective activation or repression of the targeted genes. Despite these advances, the efficient in vivo delivery of these molecules into the target cancer cells represents a critical barrier to accomplishing translation into a clinical therapy setting for cancer. Major obstacles include the large size of dCas9 fusion proteins, the necessity of multimodal delivery of protein and gRNAs, and the potential of these formulations to elicit detrimental immune responses.In this context, viral methods for delivering CRISPR face several limitations, such as the packaging capacity of the viral genome, the potential for integration of the nucleic acids into the host cells genome, and immunogenicity of viral proteins, posing serious safety concerns. The rapid development of mRNA vaccines in response to the COVID-19 pandemic has rekindled interest in mRNA-based approaches for CRISPR/dCas9 delivery. Simultaneously, due to their high loading capacity, scalability, customizable surface modification for cell targeting, and low immunogenicity, lipid nanoparticles (LNPs) have been widely explored as nonviral vectors. In this chapter, we first describe the design of optimized dCas9-effector mRNAs and gRNAs for epigenetic editing. We outline formulations of LNPs suitable for dCas9 mRNA delivery. Additionally, we provide a protocol for the co-encapsulation of the dCas9-effector mRNAs and gRNA into these LNPs, along with detailed methods for delivering these formulations to both cell lines (in vitro) and mouse models of breast cancer (in vivo).
Asunto(s)
Sistemas CRISPR-Cas , Edición Génica , Nanopartículas , Neoplasias , Edición Génica/métodos , Humanos , Nanopartículas/química , Animales , Neoplasias/genética , Neoplasias/terapia , Epigénesis Genética , Ratones , ARN Guía de Sistemas CRISPR-Cas/genética , Liposomas/química , Línea Celular Tumoral , Lípidos/química , Proteína 9 Asociada a CRISPR/genética , Proteína 9 Asociada a CRISPR/metabolismo , Terapia Genética/métodos , Técnicas de Transferencia de GenRESUMEN
Epigenetic modifications play a crucial role in regulating gene expression patterns. Through epigenetic editing approaches, the chromatin structure is modified and the activity of the targeted gene can be reprogrammed without altering the DNA sequence. By using the CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic repeats) platform with nuclease-deactivated dCas9 proteins to direct epigenetic effector domains (EDs) to genomic regulatory regions, the expression of the targeted gene can be modulated. However, the long-term stability of these effects, although demonstrated, remains unpredictable. The versatility and flexibility of (co-)targeting different genes with multiple epigenetic effectors has made the CRISPR/dCas9 platform the most widely used gene modulating technology currently available. Efficient delivery of large dCas9-ED fusion constructs into target cells, however, is challenging. An approach to overcome this limitation is to generate cells that stably express sgRNA(s) or dCas9-ED constructs. The sgRNA(s) or dCas9-ED stable cell lines can be used to study the mechanisms underlying sustained gene expression reprogramming by transiently expressing the other of the two constructs. Here, we describe a detailed protocol for the engineering of cells that stably express CRISPR/dCas9 or sgRNA. Creating a system where one component of the CRISPR/dCas9 is stably expressed while the other is transiently expressed offers a versatile platform for investigating the dynamics of epigenetic reprogramming.
Asunto(s)
Sistemas CRISPR-Cas , Epigénesis Genética , Edición Génica , ARN Guía de Sistemas CRISPR-Cas , Edición Génica/métodos , Humanos , ARN Guía de Sistemas CRISPR-Cas/genética , Línea Celular , Proteína 9 Asociada a CRISPR/metabolismo , Proteína 9 Asociada a CRISPR/genética , Células HEK293RESUMEN
Timely screening for harmful pathogens is a great challenge in emergencies where traditional culture methods suffer from long assay time and alternative methods are limited by poor accuracy and low robustness. Herein, we present a dCas9-mediated colorimetric and surface-enhanced Raman scattering (SERS) dual-signal platform (dCas9-CSD) to address this challenge. Strategically, the platform used dCas9 to accurately recognize the repetitive sequences in amplicons produced by loop-mediated isothermal amplification (LAMP), forming nucleic acid frameworks that assemble numerous bifunctional gold-platinum (Au@Pt) nanozymes into chains on the surface of streptavidin-magnetic beads (SA-MB). The collected Au@Pt converted colorless 3,3',5,5'-tetramethylbenzidine (TMB) to blue oxidized TMB (oxTMB) via its Pt shell and then enhanced the Raman signal of oxTMB by its Au core. Therefore, the presence of Salmonella could be dexterously converted into cross-validated colorimetric and SERS signals, providing more reliable conclusions. Notably, dCas9-mediated secondary recognition of amplicons reduced background signal caused by nontarget amplification, and two-round signal amplification consisting of LAMP reaction and Au@Pt catalysis greatly improved the sensitivity. With this design, Salmonella as low as 1 CFU/mL could be detected within 50 min by colorimetric and SERS modes. The robustness of dCas9-CSD was further confirmed by various real samples such as lake water, cabbage, milk, orange juice, beer, and eggs. This work provides a promising point-of-need tool for pathogen detection.
Asunto(s)
Colorimetría , Oro , Técnicas de Diagnóstico Molecular , Platino (Metal) , Bencidinas/química , Proteína 9 Asociada a CRISPR/metabolismo , Sistemas CRISPR-Cas , Oro/química , Límite de Detección , Nanopartículas del Metal/química , Técnicas de Amplificación de Ácido Nucleico , Platino (Metal)/química , Salmonella/aislamiento & purificación , Salmonella/genética , Espectrometría RamanRESUMEN
Eubacterium limosum is a Clostridial acetogen that efficiently utilizes a wide range of single-carbon substrates and contributes to metabolism of health-associated compounds in the human gut microbiota. These traits have led to interest in developing it as a platform for sustainable CO2-based biofuel production to combat carbon emissions, and for exploring the importance of the microbiota in human health. However, synthetic biology and metabolic engineering in E. limosum have been hindered by the inability to rapidly make precise genomic modifications. Here, we screened a diverse library of recombinase proteins to develop a highly efficient oligonucleotide-based recombineering system based on the viral recombinase RecT. Following optimization, the system is capable of catalyzing ssDNA recombination at an efficiency of up to 2%. Addition of a Cas9 counterselection system eliminated unrecombined cells, with up to 100% of viable cells encoding the desired mutation, enabling creation of genomic point mutations in a scarless and markerless manner. We deployed this system to create a clean knockout of the extracellular polymeric substance (EPS) gene cluster, generating a strain incapable of biofilm formation. This approach is rapid and simple, not requiring laborious homology arm cloning, and can readily be retargeted to almost any genomic locus. This work overcomes a major bottleneck in E. limosum genetic engineering by enabling precise genomic modifications, and provides both a roadmap and associated recombinase plasmid library for developing similar systems in other Clostridia of interest.
Asunto(s)
Sistemas CRISPR-Cas , Eubacterium , Eubacterium/genética , Sistemas CRISPR-Cas/genética , Ingeniería Metabólica/métodos , Recombinación Genética/genética , Genoma Bacteriano/genética , Proteína 9 Asociada a CRISPR/genética , Proteína 9 Asociada a CRISPR/metabolismo , Recombinasas/genética , Recombinasas/metabolismo , Ingeniería Genética/métodos , Edición Génica/métodos , Familia de MultigenesRESUMEN
The efficiency and outcome of CRISPR/Cas9 editing depends on the chromatin state at the cut site. It has been shown that changing the chromatin state can influence both the efficiency and repair outcome, and epigenetic drugs have been used to improve Cas9 editing. However, because the target proteins of these drugs are not homogeneously distributed across the genome, the efficacy of these drugs may be expected to vary from locus to locus. Here, we systematically analyzed this chromatin context-dependency for 160 epigenetic drugs. We used a human cell line with 19 stably integrated reporters to induce a double-stranded break in different chromatin environments. We then measured Cas9 editing efficiency and repair pathway usage by sequencing the mutational signatures. We identified 58 drugs that modulate Cas9 editing efficiency and/or repair outcome dependent on the local chromatin environment. For example, we find a subset of histone deacetylase inhibitors that improve Cas9 editing efficiency throughout all types of heterochromatin (e.g. PCI-24781), while others were only effective in euchromatin and H3K27me3-marked regions (e.g. apicidin). In summary, this study reveals that most epigenetic drugs alter CRISPR editing in a chromatin-dependent manner, and provides a resource to improve Cas9 editing more selectively at the desired location.
Asunto(s)
Sistemas CRISPR-Cas , Cromatina , Epigénesis Genética , Edición Génica , Inhibidores de Histona Desacetilasas , Humanos , Edición Génica/métodos , Epigénesis Genética/efectos de los fármacos , Cromatina/metabolismo , Cromatina/genética , Inhibidores de Histona Desacetilasas/farmacología , Reparación del ADN , Proteína 9 Asociada a CRISPR/metabolismo , Proteína 9 Asociada a CRISPR/genética , Heterocromatina/metabolismo , Heterocromatina/genética , Línea Celular , Histonas/metabolismo , Eucromatina/genética , Roturas del ADN de Doble Cadena/efectos de los fármacosRESUMEN
DNA base editing technologies predominantly utilize engineered deaminases, limiting their ability to edit thymine and guanine directly. In this study, we successfully achieve base editing of both cytidine and thymine by leveraging the translesion DNA synthesis pathway through the engineering of uracil-DNA glycosylase (UNG). Employing structure-based rational design, exploration of homologous proteins, and mutation screening, we identify a Deinococcus radiodurans UNG mutant capable of effectively editing thymine. When fused with the nickase Cas9, the engineered DrUNG protein facilitates efficient thymine base editing at endogenous sites, achieving editing efficiencies up to 55% without enrichment and exhibiting minimal cellular toxicity. This thymine base editor (TBE) exhibits high editing specificity and significantly restores IDUA enzyme activity in cells derived from patients with Hurler syndrome. TBEs represent efficient, specific, and low-toxicity approaches to base editing with potential applications in treating relevant diseases.
Asunto(s)
Edición Génica , Uracil-ADN Glicosidasa , Uracil-ADN Glicosidasa/metabolismo , Uracil-ADN Glicosidasa/genética , Edición Génica/métodos , Humanos , Ingeniería de Proteínas/métodos , ADN/metabolismo , ADN/genética , Timina/metabolismo , Deinococcus/genética , Deinococcus/enzimología , Deinococcus/metabolismo , Proteína 9 Asociada a CRISPR/metabolismo , Proteína 9 Asociada a CRISPR/genética , Mutación , Células HEK293 , Sistemas CRISPR-CasRESUMEN
RNA serves as information media as well as molecular scaffold in nature and synthetic systems. The single guide RNA (sgRNA) widely applied in CRISPR techniques exemplifies both functions, with a guide region bearing DNA base-pairing information, and a structural motif for Cas9 protein scaffolding. The scaffold region has been modified by fusing RNA aptamers to the tetra-stem loop. The guide region is typically not regarded as a pluggable module as it encodes the essential function of DNA sequence recognition. Here, we investigate a chimera of two sgRNAs, with distinct guide sequences joined by an RNA linker (dgRNA), regarding its DNA binding function and loop induction capability. First, we studied the sequence bi-specificity of the dgRNA and discovered that the RNA linker allows distal parts of double-stranded DNA to be brought into proximity. To test the activity of the dgRNA in organisms, we used the LacZ gene as a reporter and recapitulated the loop-mediated gene inhibition by LacI in E. coli. We found that the dgRNA can be applied to target distal genomic regions with comparable levels of inhibition. The capability of dgRNA to induce DNA contacts solely requires dCas9 and RNA, making it a minimal system to remodel chromosomal conformation in various organisms.
Asunto(s)
Escherichia coli , ARN Guía de Sistemas CRISPR-Cas , Escherichia coli/genética , Escherichia coli/metabolismo , ARN Guía de Sistemas CRISPR-Cas/genética , ARN Guía de Sistemas CRISPR-Cas/metabolismo , Conformación de Ácido Nucleico , Sistemas CRISPR-Cas , ADN/metabolismo , ADN/química , ADN/genética , ADN Bacteriano/metabolismo , ADN Bacteriano/genética , Aptámeros de Nucleótidos/química , Aptámeros de Nucleótidos/genética , Aptámeros de Nucleótidos/metabolismo , Proteína 9 Asociada a CRISPR/metabolismo , Proteína 9 Asociada a CRISPR/genéticaRESUMEN
Gene/segmental duplications play crucial roles in genome evolution and variation. Here, we introduce paired nicking-induced amplification (PNAmp) for their experimental induction. PNAmp strategically places two Cas9 nickases upstream and downstream of a replication origin on opposite strands. This configuration directs the sister replication forks initiated from the origin to break at the nicks, generating a pair of one-ended double-strand breaks. If homologous sequences flank the two break sites, then end resection converts them to single-stranded DNAs that readily anneal to drive duplication of the region bounded by the homologous sequences. PNAmp induces duplication of segments as large as â¼1 Mb with efficiencies exceeding 10% in the budding yeast Saccharomyces cerevisiae. Furthermore, appropriate splint DNAs allow PNAmp to duplicate/multiplicate even segments not bounded by homologous sequences. We also provide evidence for PNAmp in mammalian cells. Therefore, PNAmp provides a prototype method to induce structural variations by manipulating replication fork progression.
Asunto(s)
Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Humanos , Replicación del ADN , Proteína 9 Asociada a CRISPR/genética , Proteína 9 Asociada a CRISPR/metabolismo , Duplicación de Gen , Origen de Réplica/genética , Roturas del ADN de Doble Cadena , Sistemas CRISPR-Cas/genéticaRESUMEN
The RNA-guided DNA endonuclease Cas9 cleaves double-stranded DNA targets complementary to an RNA guide, and is widely used as a powerful genome-editing tool. Here, we report the crystal structure of Brevibacillus laterosporus Cas9 (BlCas9, also known as BlatCas9), in complex with a guide RNA and its target DNA at 2.4-Å resolution. The structure reveals that the BlCas9 guide RNA adopts an unexpected architecture containing a triple-helix, which is specifically recognized by BlCas9, and that BlCas9 recognizes a unique N4CNDN protospacer adjacent motif through base-specific interactions on both the target and non-target DNA strands. Based on the structure, we rationally engineered a BlCas9 variant that exhibits enhanced genome- and base-editing activities with an expanded target scope in human cells. This approach may further improve the performance of the enhanced BlCas9 variant to generate useful genome-editing tools that require only a single C PAM nucleotide and can be packaged into a single AAV vector for in vivo gene therapy.
Asunto(s)
Brevibacillus , Proteína 9 Asociada a CRISPR , Edición Génica , Brevibacillus/genética , Brevibacillus/metabolismo , Brevibacillus/enzimología , Edición Génica/métodos , Proteína 9 Asociada a CRISPR/genética , Proteína 9 Asociada a CRISPR/metabolismo , Proteína 9 Asociada a CRISPR/química , ARN Guía de Sistemas CRISPR-Cas/genética , ARN Guía de Sistemas CRISPR-Cas/metabolismo , Humanos , Sistemas CRISPR-Cas , Ingeniería de Proteínas/métodosRESUMEN
The outcome of CRISPR-Cas-mediated genome modifications is dependent on DNA double-strand break (DSB) processing and repair pathway choice. Homology-directed repair (HDR) of protein-blocked DSBs requires DNA end resection that is initiated by the endonuclease activity of the MRE11 complex. Using reconstituted reactions, we show that Cas9 breaks are unexpectedly not directly resectable by the MRE11 complex. In contrast, breaks catalyzed by Cas12a are readily processed. Cas9, unlike Cas12a, bridges the broken ends, preventing DSB detection and processing by MRE11. We demonstrate that Cas9 must be dislocated after DNA cleavage to allow DNA end resection and repair. Using single molecule and bulk biochemical assays, we next find that the HLTF translocase directly removes Cas9 from broken ends, which allows DSB processing by DNA end resection or non-homologous end-joining machineries. Mechanistically, the activity of HLTF requires its HIRAN domain and the release of the 3'-end generated by the cleavage of the non-target DNA strand by the Cas9 RuvC domain. Consequently, HLTF removes the H840A but not the D10A Cas9 nickase. The removal of Cas9 H840A by HLTF explains the different cellular impact of the two Cas9 nickase variants in human cells, with potential implications for gene editing.
Asunto(s)
Proteína 9 Asociada a CRISPR , Sistemas CRISPR-Cas , Roturas del ADN de Doble Cadena , ADN , Humanos , Proteína 9 Asociada a CRISPR/metabolismo , Proteína 9 Asociada a CRISPR/genética , ADN/metabolismo , ADN/genética , Proteína Homóloga de MRE11/metabolismo , Proteína Homóloga de MRE11/genética , Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al ADN/genética , Proteínas Asociadas a CRISPR/metabolismo , Proteínas Asociadas a CRISPR/genética , Edición Génica , Endonucleasas/metabolismo , Endonucleasas/genética , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Endodesoxirribonucleasas/metabolismo , Endodesoxirribonucleasas/genética , Reparación del ADN por Unión de Extremidades , División del ADN , Factores de Transcripción/metabolismo , Factores de Transcripción/genéticaRESUMEN
The contraction of CAG/CTG repeats is an attractive approach to correct the mutation that causes at least 15 neuromuscular and neurodegenerative diseases, including Huntington's disease and Myotonic Dystrophy type 1. Contractions can be achieved in vivo using the Cas9 D10A nickase from Streptococcus pyogenes (SpCas9) using a single guide RNA (sgRNA) against the repeat tract. One hurdle on the path to the clinic is that SpCas9 is too large to be packaged together with its sgRNA into a single adeno-associated virus. Here we aimed to circumvent this problem using the smaller Cas9 orthologue, SlugCas9, and the Cas9 ancestor OgeuIscB. We found them to be ineffective in inducing contractions, despite their advertised PAM sequences being compatible with CAG/CTG repeats. Thus, we further developed smaller Cas9 hybrids, made of the PAM interacting domain of S. pyogenes and the catalytic domains of the smaller Cas9 orthologues. We also designed the cognate sgRNA hybrids using molecular dynamic simulations and binding energy calculations. We found that the four Cas9/sgRNA hybrid pairs tested in human cells failed to edit their target sequences. We conclude that in silico approaches can identify functional changes caused by point mutations but are not sufficient for designing larger scale complexes of Cas9/sgRNA hybrids.