RESUMEN
With the rapid development of CRISPR-Cas9 technology, gene editing has become a powerful tool for studying gene function. Specifically, in the study of the mechanisms by which natural immune responses combat viral infections, gene knockout mouse models have provided an indispensable platform. This article describes a detailed protocol for constructing gene knockout mice using the CRISPR-Cas9 system. This field focuses on the design of single-guide RNAs (sgRNAs) targeting the antiviral immune gene cGAS, embryo microinjection, and screening and verification of gene editing outcomes. Furthermore, this study provides methods for using cGAS gene knockout mice to analyze the role of specific genes in natural immune responses. Through this protocol, researchers can efficiently generate specific gene knockout mouse models, which not only helps in understanding the functions of the immune system but also offers a powerful experimental tool for exploring the mechanisms of antiviral innate immunity.
Asunto(s)
Sistemas CRISPR-Cas , Edición Génica , Inmunidad Innata , Ratones Noqueados , ARN Guía de Sistemas CRISPR-Cas , Animales , Inmunidad Innata/genética , Ratones , ARN Guía de Sistemas CRISPR-Cas/genética , Edición Génica/métodos , Técnicas de Inactivación de Genes/métodos , Nucleotidiltransferasas/genética , Nucleotidiltransferasas/metabolismo , Virosis/inmunología , Virosis/genéticaRESUMEN
Realizing the full potential of genome editing for crop improvement has been slow due to inefficient methods for reagent delivery and the reliance on tissue culture for creating gene-edited plants. RNA viral vectors offer an alternative approach for delivering gene engineering reagents and bypassing the tissue culture requirement. Viruses, however, are often excluded from the shoot apical meristem, making virus-mediated gene editing inefficient in some species. Here, we developed effective approaches for generating gene-edited shoots in Cas9-expressing transgenic tomato plants using RNA virus-mediated delivery of single-guide RNAs (sgRNAs). RNA viral vectors expressing sgRNAs were either delivered to leaves or sites near axillary meristems. Trimming of the apical and axillary meristems induced new shoots to form from edited somatic cells. To further encourage the induction of shoots, we used RNA viral vectors to deliver sgRNAs along with the cytokinin biosynthesis gene, isopentenyl transferase. Abundant, phenotypically normal, gene-edited shoots were induced per infected plant with single and multiplexed gene edits fixed in the germline. The use of viruses to deliver both gene editing reagents and developmental regulators overcomes the bottleneck in applying virus-induced gene editing to dicotyledonous crops such as tomato and reduces the dependency on tissue culture.
Asunto(s)
Edición Génica , Meristema , Plantas Modificadas Genéticamente , ARN Guía de Sistemas CRISPR-Cas , Solanum lycopersicum , Solanum lycopersicum/genética , Edición Génica/métodos , Meristema/genética , ARN Guía de Sistemas CRISPR-Cas/genética , ARN Guía de Sistemas CRISPR-Cas/metabolismo , Vectores Genéticos/genética , Sistemas CRISPR-Cas , Brotes de la Planta/genética , Brotes de la Planta/virología , Virus ARN/genética , Transferasas Alquil y ArilRESUMEN
CRISPR homing gene drives can suppress pest populations by targeting female fertility genes, converting wild-type alleles into drive alleles in the germline of drive heterozygotes. fsRIDL (female-specific Release of Insects carrying a Dominant Lethal) is a self-limiting population suppression strategy involving continual release of transgenic males carrying female lethal alleles. Here, we propose an improved pest suppression system called "Release of Insects carrying a Dominant-sterile Drive" (RIDD), combining performance characteristics of homing drive and fsRIDL. We construct a split RIDD system in Drosophila melanogaster by creating a 3-gRNA drive disrupting the doublesex female exon. Drive alleles bias their inheritance in males, while drive alleles and resistance alleles formed by end-joining cause dominant female sterility. Weekly releases of RIDD males progressively suppressed and eventually eliminated cage populations. Modeling shows that RIDD is substantially stronger than SIT and fsRIDL. RIDD is also self-limiting, potentially allowing targeted population suppression.
Asunto(s)
Animales Modificados Genéticamente , Proteínas de Drosophila , Drosophila melanogaster , Tecnología de Genética Dirigida , Animales , Femenino , Masculino , Drosophila melanogaster/genética , Tecnología de Genética Dirigida/métodos , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Alelos , Sistemas CRISPR-Cas , Genes Dominantes , Control Biológico de Vectores/métodos , Infertilidad/genética , Infertilidad/terapia , ARN Guía de Sistemas CRISPR-Cas/genética , Proteínas de Unión al ADNRESUMEN
CRISPR is a precise and effective genome editing technology; but despite several advancements during the last decade, our ability to computationally design gRNAs remains limited. Most predictive models have relatively low predictive power and utilize only the sequence of the target site as input. Here we suggest a new category of features, which incorporate the target site genomic position and the presence of genes close to it. We calculate four features based on gene expression and codon usage bias indices. We show, on CRISPR datasets taken from 3 different cell types, that such features perform comparably with 425 state-of-the-art predictive features, ranking in the top 2-12% of features. We trained new predictive models, showing that adding expression features to them significantly improves their r2 by up to 0.04 (relative increase of 39%), achieving average correlations of up to 0.38 on their validation sets; and that these features are deemed important by different feature importance metrics. We believe that incorporating the target site's position, in addition to its sequence, in features such as we have generated here will improve our ability to predict, design and understand CRISPR experiments going forward.
Asunto(s)
Sistemas CRISPR-Cas , Uso de Codones , Edición Génica , Uso de Codones/genética , Edición Génica/métodos , Sistemas CRISPR-Cas/genética , Humanos , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/genética , Biología Computacional/métodos , ARN Guía de Sistemas CRISPR-Cas/genética , Codón/genética , Expresión Génica/genéticaRESUMEN
As a widely used eukaryotic model organism, Neurospora crassa offers advantages in genetic studies due to its diverse biology and rapid growth. Traditional genetic manipulation methods, such as homologous recombination, require a considerable amount of time and effort. In this study, we present an easy-to-use CRIPSR/Cas9 system for N. crassa, in which the cas9 sequence is incorporated into the fungal genome and naked guide RNA is introduced via electroporation. Our approach eliminates the need for constructing multiple vectors, speeding up the mutagenesis process. Using cyclosporin-resistant-1 (csr-1) as a selectable marker gene, we achieved 100% editing efficiency under selection conditions. Furthermore, we successfully edited the non-selectable gene N-acylethanolamine amidohydrolase-2 (naa-2), demonstrating the versatility of the system. Combining gRNAs targeting csr-1 and naa-2 simultaneously increased the probability of finding mutants carrying the non-selectable mutation. The system is not only user-friendly but also effective, providing a rapid and efficient method for generating loss-of-function mutants in N. crassa compared to traditional methods.
Asunto(s)
Sistemas CRISPR-Cas , Edición Génica , Mutagénesis , Neurospora crassa , Neurospora crassa/genética , Edición Génica/métodos , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , ARN Guía de Sistemas CRISPR-Cas/genética , Genoma FúngicoRESUMEN
BACKGROUND: The type II based CRISPR-Cas system remains restrictedly utilized in archaea, a featured domain of life that ranks parallelly with Bacteria and Eukaryotes. Methanococcus maripaludis, known for rapid growth and genetic tractability, serves as an exemplary model for studying archaeal biology and exploring CO2-based biotechnological applications. However, tools for controlled gene regulation remain deficient and CRISPR-Cas tools still need improved in this archaeon, limiting its application as an archaeal model cellular factory. RESULTS: This study not only improved the CRISPR-Cas9 system for optimizing multiplex genome editing and CRISPR plasmid construction efficiencies but also pioneered an effective CRISPR interference (CRISPRi) system for controlled gene regulation in M. maripaludis. We developed two novel strategies for balanced expression of multiple sgRNAs, facilitating efficient multiplex genome editing. We also engineered a strain expressing Cas9 genomically, which simplified the CRISPR plasmid construction and facilitated more efficient genome modifications, including markerless and scarless gene knock-in. Importantly, we established a CRISPRi system using catalytic inactive dCas9, achieving up to 100-fold repression on target gene. Here, sgRNAs targeting near and downstream regions of the transcription start site and the 5'end ORF achieved the highest repression efficacy. Furthermore, we developed an inducible CRISPRi-dCas9 system based on TetR/tetO platform. This facilitated the inducible gene repression, especially for essential genes. CONCLUSIONS: Therefore, these advancements not only expand the toolkit for genetic manipulation but also bridge methodological gaps for controlled gene regulation, especially for essential genes, in M. maripaludis. The robust toolkit developed here paves the way for applying M. maripaludis as a vital model archaeal cell factory, facilitating fundamental biological studies and applied biotechnology development of archaea.
Asunto(s)
Sistemas CRISPR-Cas , Edición Génica , Methanococcus , Methanococcus/genética , Edición Génica/métodos , Plásmidos/genética , ARN Guía de Sistemas CRISPR-Cas/genética , Genoma Arqueal , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Ingeniería Genética/métodosRESUMEN
In addressing the limitations of CRISPR-Cas9, including off-target effects and high licensing fees for commercial use, Cas-CLOVER, a dimeric gene editing tool activated by two guide RNAs, was recently developed. This study focused on implementing and evaluating Cas-CLOVER in HEK-293 cells used for recombinant adeno-associated virus (rAAV) production by targeting the signal transducer and activator of transcription 1 (STAT1) locus, which is crucial for cell growth regulation and might influence rAAV production yields. Cas-CLOVER demonstrated impressive efficiency in gene editing, achieving over 90% knockout (KO) success. Thirteen selected HEK-293 STAT1 KO sub-clones were subjected to extensive analytical characterization to assess their genomic stability, crucial for maintaining cell integrity and functionality. Additionally, rAAV9 productivity, Rep protein pattern profile, and potency, among others, were assessed. Clones showed significant variation in capsid and vector genome titers, with capsid titer reductions ranging from 15% to 98% and vector genome titers from 16% to 55%. Interestingly, the Cas-CLOVER-mediated STAT1 KO bulk cell population showed a better ratio of full to empty capsids. Our study also established a comprehensive analytical workflow to detect and evaluate the gene KOs generated by this innovative tool, providing a solid groundwork for future research in precise gene editing technologies.
Asunto(s)
Sistemas CRISPR-Cas , Dependovirus , Edición Génica , Técnicas de Inactivación de Genes , Factor de Transcripción STAT1 , Humanos , Dependovirus/genética , Factor de Transcripción STAT1/genética , Factor de Transcripción STAT1/metabolismo , Células HEK293 , Sistemas CRISPR-Cas/genética , Edición Génica/métodos , Técnicas de Inactivación de Genes/métodos , Vectores Genéticos/genética , ARN Guía de Sistemas CRISPR-Cas/genéticaRESUMEN
Duchenne Muscular dystrophy (DMD), a yet-incurable X-linked recessive disorder that results in muscle wasting and loss of ambulation is due to mutations in the dystrophin gene. Exonic duplications of dystrophin gene are a common type of mutations found in DMD patients. In this study, we utilized a single guide RNA CRISPR strategy targeting intronic regions to delete the extra duplicated regions in patient myogenic cells carrying duplication of exon 2, exons 2-9, and exons 8-9 in the DMD gene. Immunostaining on CRISPR-corrected derived myotubes demonstrated the rescue of dystrophin protein. Subsequent RNA sequencing of the DMD cells indicated rescue of genes of dystrophin related pathways. Examination of predicted close-match off-targets evidenced no aberrant gene editing at these loci. Here, we further demonstrate the efficiency of a single guide CRISPR strategy capable of deleting multi-exon duplications in the DMD gene without significant off target effect. Our study contributes valuable insights into the safety and efficacy of using single guide CRISPR strategy as a potential therapeutic approach for DMD patients with duplications of variable size.
Asunto(s)
Sistemas CRISPR-Cas , Distrofina , Exones , Duplicación de Gen , Edición Génica , Distrofia Muscular de Duchenne , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/terapia , Humanos , Exones/genética , Distrofina/genética , Edición Génica/métodos , ARN Guía de Sistemas CRISPR-Cas/genética , Terapia Genética/métodos , Fibras Musculares Esqueléticas/metabolismoRESUMEN
In recent years, various technologies have emerged for the imaging of chromatin loci in living cells via catalytically inactive Cas9 (dCas9). These technologies facilitate a deeper understanding of the mechanisms behind the chromatin dynamics and provide valuable kinetic data that could not have previously been obtained via FISH applied to fixed cells. However, such technologies are relatively complicated, as they involve the expression of several chimeric proteins as well as sgRNAs targeting the visualized loci, a process that entails many technical subtleties. Therefore, the effectiveness in visualizing a specific target locus may be quite low. In this study, we directly compared two versions of a previously published CRISPR-Sirius method based on the use of sgRNAs containing eight MS2 or PP7 stem loops and the expression of MCP or PCP fused to fluorescent proteins. We assessed the visualization efficiency for several unique genomic loci by comparing the two approaches in delivering sgRNA genes (transient transfection and lentiviral transduction), as well as two CRISPR-Sirius versions (with PCP and with MCP). The efficiency of visualization varied among the loci, and not all loci could be visualized. However, the MCP-sfGFP version provided more efficient visualization in terms of the number of cells with signals than PCP-sfGFP for all tested loci. We also showed that lentiviral transduction was more efficient in locus imaging than transient transfection for both CRISPR-Sirius systems. Most of the target loci in our study were located at the borders of topologically associating domains, and we defined a set of TAD borders that could be effectively visualized using the MCP-sfGFP version of the CRISPR-Sirius system. Altogether, our study validates the use of the CRISPR-Sirius technology for live-cell visualization and highlights various technical details that should be considered when using this method.
Asunto(s)
Sistemas CRISPR-Cas , Humanos , Sistemas CRISPR-Cas/genética , Cromatina/metabolismo , Cromatina/genética , Células HEK293 , ARN Guía de Sistemas CRISPR-Cas/genética , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/genéticaRESUMEN
MOTIVATION: Pooled single-cell CRISPR screens have emerged as a powerful tool in functional genomics to probe the effect of genetic interventions at scale. A crucial step in the analysis of the resulting data is the assignment of cells to gRNAs corresponding to a specific genetic intervention. However, this step is challenging due to a lack of systematic benchmarks and accessible software to apply and compare different guide assignment strategies. To address this, we here propose crispat (CRISPR guide assignment tool), a Python package to facilitate the choice of a suitable guide assignment strategy for single-cell CRISPR screens. RESULTS: We demonstrate the package on four single-cell CRISPR interference screens at low multiplicity of infection from two studies, where crispat identifies strong differences in the number of assigned cells, downregulation of the target genes and number of discoveries across different guide assignment strategies, highlighting the need for a suitable guide assignment strategy to obtain optimal power in single-cell CRISPR screens. AVAILABILITY AND IMPLEMENTATION: crispat is implemented in python, the source code, installation instructions and tutorials can be found at https://github.com/velten-group/crispat and it can be installed from PyPI (https://pypi.org/project/crispat/). Code to reproduce all findings in this paper is available at https://github.com/velten-group/crispat_analysis, as well as at https://zenodo.org/records/13373265.
Asunto(s)
Sistemas CRISPR-Cas , ARN Guía de Sistemas CRISPR-Cas , Análisis de la Célula Individual , Programas Informáticos , Análisis de la Célula Individual/métodos , ARN Guía de Sistemas CRISPR-Cas/genética , Humanos , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente EspaciadasRESUMEN
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
In recent years, targeted genome editing has emerged as an indispensable tool for creating animal models, facilitating a comprehensive exploration of the molecular mechanisms governing a myriad of biological processes. Within this scientific landscape, the investigation of meiosis in mice has attracted considerable attention across numerous research laboratories. The precision and versatility of the CRISPR/Cas9 genome editing system have revolutionized our ability to generate mice with tailored genetic alterations, including point mutations and null mutations. These genetic modifications have provided invaluable insights into the intricate functionality of various meiotic genes and their associated variants. In this context, we present a detailed state of the art protocol for the creation of novel mouse models, each bearing specific genetic modifications within key meiotic genes, through the application of CRISPR/Cas9 technology. Furthermore, we showcase two distinct genetic modifications, accomplished within our laboratory, that can serve as valuable reference points for researchers seeking to elucidate the molecular intricacies of meiosis in mammals.
Asunto(s)
Sistemas CRISPR-Cas , Edición Génica , Meiosis , Animales , Meiosis/genética , Ratones , Edición Génica/métodos , Masculino , Modelos Animales , Femenino , ARN Guía de Sistemas CRISPR-Cas/genéticaRESUMEN
An electrochemical sensor assisted by primer exchange reaction (PER) and CRISPR/Cas9 system (PER-CRISPR/Cas9-E) was established for the sensitive detection of dual microRNAs (miRNAs). Two PER hairpin (HP) were designed to produce a lot of extended PER products, which could hybridize with two kinds of hairpin probes modified on the electrode and initiate the cleavage of two CRISPR/Cas9 systems guided by single guide RNAs (sgRNAs) with different recognition sequences. The decrease of the two electrochemical redox signals indicated the presence of dual-target miRNAs. With the robustness and high specificity of PER amplification and CRISPR/Cas9 cleavage system, simultaneous detection of two targets was achieved and the detection limits for miRNA-21 and miRNA-155 were 0.43 fM and 0.12 fM, respectively. The developed biosensor has the advantages of low cost, easy operation, and in-situ detection, providing a promising platform for point-of-care detection of multiple miRNAs.
Asunto(s)
Técnicas Biosensibles , Sistemas CRISPR-Cas , Técnicas Electroquímicas , Límite de Detección , MicroARNs , MicroARNs/análisis , MicroARNs/genética , Sistemas CRISPR-Cas/genética , Técnicas Electroquímicas/métodos , Técnicas Biosensibles/métodos , Humanos , ARN Guía de Sistemas CRISPR-Cas/genéticaRESUMEN
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
CRISPR/Cas-based transcriptional activators can be enhanced by intrinsically disordered regions (IDRs). However, the underlying mechanisms are still debatable. Here, we examine 12 well-known IDRs by fusing them to the dCas9-VP64 activator, of which only seven can augment activation, albeit independently of their phase separation capabilities. Moreover, modular domains (MDs), another class of multivalent molecules, though ineffective in enhancing dCas9-VP64 activity on their own, show substantial enhancement in transcriptional activation when combined with dCas9-VP64-IDR. By varying the number of gRNA binding sites and fusing dCas9-VP64 with different IDRs/MDs, we uncover that optimal, rather than maximal, cis-trans cooperativity enables the most robust activation. Finally, targeting promoter-enhancer pairs yields synergistic effects, which can be further amplified via enhancing chromatin interactions. Overall, our study develops a versatile platform for efficient gene activation and sheds important insights into CRIPSR-based transcriptional activators enhanced with multivalent molecules.
Asunto(s)
Sistemas CRISPR-Cas , Activación Transcripcional , Humanos , Regiones Promotoras Genéticas , ARN Guía de Sistemas CRISPR-Cas/genética , ARN Guía de Sistemas CRISPR-Cas/metabolismo , Células HEK293 , Sitios de Unión , Cromatina/metabolismo , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Elementos de Facilitación GenéticosRESUMEN
The invention of next-generation CRISPR/Cas gene editing tools, like base and prime editing, for correction of gene variants causing disease, has created hope for in vivo use in patients leading to wider clinical translation. To realize this potential, delivery vehicles that can ferry gene editing tool kits safely and effectively into specific cell populations or tissues are in great demand. In this review, we describe the development of enveloped retrovirus-derived particles as carriers of "ready-to-work" ribonucleoprotein complexes consisting of Cas9-derived editor proteins and single guide RNAs. We present arguments for adapting viruses for cell-targeted protein delivery and describe the status after a decade-long development period, which has already shown effective editing in primary cells, including T cells and hematopoietic stem cells, and in tissues targeted in vivo, including mouse retina, liver, and brain. Emerging evidence has demonstrated that engineered virus-derived nanoparticles can accommodate both base and prime editors and seems to fertilize a sprouting hope that such particles can be further developed and produced in large scale for therapeutic applications.
Asunto(s)
Sistemas CRISPR-Cas , Edición Génica , Ribonucleoproteínas , Edición Génica/métodos , Humanos , Ribonucleoproteínas/metabolismo , Ribonucleoproteínas/genética , Animales , Virión/metabolismo , Virión/genética , Retroviridae/genética , Vectores Genéticos/genética , Terapia Genética/métodos , Técnicas de Transferencia de Gen , ARN Guía de Sistemas CRISPR-Cas/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
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.