RÉSUMÉ
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.
Sujet(s)
RNA polymerase II , Transcription génétique , Globines bêta , Humains , RNA polymerase II/métabolisme , RNA polymerase II/génétique , Globines bêta/génétique , Globines bêta/métabolisme , ADN/métabolisme , ADN/génétique , Régions promotrices (génétique) , Protéine-9 associée à CRISPR/métabolisme , Protéine-9 associée à CRISPR/génétique , Systèmes CRISPR-Cas , ARN/génétique , ARN/métabolisme , /génétique , ARN messager/génétique , ARN messager/métabolisme , Lignée cellulaireRÉSUMÉ
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.
Sujet(s)
ADN simple brin , Édition de gène , Rad51 Recombinase , Réparation de l'ADN par recombinaison , ADN simple brin/métabolisme , ADN simple brin/génétique , Humains , Édition de gène/méthodes , Rad51 Recombinase/métabolisme , Rad51 Recombinase/génétique , Systèmes CRISPR-Cas , Cellules HEK293 , Protéines associées aux CRISPR/métabolisme , Protéines associées aux CRISPR/génétique , Protéine-9 associée à CRISPR/métabolisme , Protéine-9 associée à CRISPR/génétique , Protéines bactériennes/métabolisme , Protéines bactériennes/génétique , Endodeoxyribonucleases/métabolisme , Endodeoxyribonucleases/génétique , Réparation de l'ADN par jonction d'extrémitésRÉSUMÉ
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.
Sujet(s)
Protéine-9 associée à CRISPR , Systèmes CRISPR-Cas , Édition de gène , , Streptococcus pyogenes , Édition de gène/méthodes , Protéine-9 associée à CRISPR/métabolisme , Protéine-9 associée à CRISPR/génétique , Humains , /génétique , Streptococcus pyogenes/génétique , Streptococcus pyogenes/enzymologie , Motifs nucléotidiques , Clustered regularly interspaced short palindromic repeatsRÉSUMÉ
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.
Sujet(s)
Cellules endothéliales , Édition de gène , Récepteur-2 au facteur croissance endothéliale vasculaire , Récepteur-2 au facteur croissance endothéliale vasculaire/métabolisme , Récepteur-2 au facteur croissance endothéliale vasculaire/génétique , Humains , Édition de gène/méthodes , Cellules endothéliales/métabolisme , Néovascularisation physiologique , Systèmes CRISPR-Cas , Phosphorylation , , Facteur de croissance endothéliale vasculaire de type A/métabolisme , Facteur de croissance endothéliale vasculaire de type A/génétique , Néovascularisation pathologique/métabolisme , Protéine-9 associée à CRISPR/métabolisme , Protéine-9 associée à CRISPR/génétique , Rétine/métabolisme , Vecteurs génétiques ,RÉSUMÉ
In utero gene editing (IUGE) is a potential treatment for inherited diseases that cause pathology before or soon after birth. Preexisting immunity to adeno-associated virus (AAV) vectors and Cas9 endonuclease may limit postnatal gene editing. The tolerogenic fetal immune system minimizes a fetal immune barrier to IUGE. However, the ability of maternal immunity to limit fetal gene editing remains a question. We investigated whether preexisting maternal immunity to AAV or Cas9 impairs IUGE. Using a combination of fluorescent reporter mice and a murine model of a metabolic liver disease, we demonstrated that maternal anti-AAV IgG antibodies were efficiently transferred from dam to fetus and impaired IUGE in a maternal titer-dependent fashion. By contrast, maternal cellular immunity was inefficiently transferred to the fetus, and neither maternal cellular nor humoral immunity to Cas9 impaired IUGE. Using human umbilical cord and maternal blood samples collected from mid- to late-gestation pregnancies, we demonstrated that maternal-fetal transmission of anti-AAV IgG was inefficient in midgestation compared with term, suggesting that the maternal immune barrier to clinical IUGE would be less relevant at midgestation. These findings support immunologic advantages for IUGE and inform maternal preprocedural testing protocols and exclusion criteria for future clinical trials.
Sujet(s)
Dependovirus , Édition de gène , Animaux , Femelle , Dependovirus/génétique , Dependovirus/immunologie , Souris , Grossesse , Humains , Immunoglobuline G/immunologie , Immunoglobuline G/génétique , Immunoglobuline G/sang , Protéine-9 associée à CRISPR/génétique , Protéine-9 associée à CRISPR/immunologie , Vecteurs génétiques/immunologie , Échange foetomaternel/immunologie , Échange foetomaternel/génétique , Anticorps antiviraux/immunologie , Anticorps antiviraux/sang , Systèmes CRISPR-Cas , Foetus/immunologie , Immunité acquise d'origine maternelle/immunologieRÉSUMÉ
Despite the demonstrated importance of DNA G-quadruplexes (G4s) in health and disease, technologies to readily manipulate specific G4 folding for functional analysis and therapeutic purposes are lacking. Here we employ G4-stabilizing protein/ligand in conjunction with CRISPR to selectively facilitate single or multiple targeted G4 folding within specific genomic loci. We demonstrate that fusion of nucleolin with a catalytically inactive Cas9 can specifically stabilize G4s in the promoter of oncogene MYC and muscle-associated gene Itga7 as well as telomere G4s, leading to cell proliferation arrest, inhibition of myoblast differentiation and cell senescence, respectively. Furthermore, CRISPR can confer intra-G4 selectivity to G4-binding compounds pyridodicarboxamide and pyridostatin. Compared with traditional G4 ligands, CRISPR-guided biotin-conjugated pyridodicarboxamide enables a more precise investigation into the biological functionality of de novo G4s. Our study provides insights that will enhance understanding of G4 functions and therapeutic interventions.
Sujet(s)
Systèmes CRISPR-Cas , G-quadruplexes , , Protéines de liaison à l'ARN , Humains , Ligands , Protéines de liaison à l'ARN/métabolisme , Protéines de liaison à l'ARN/génétique , Phosphoprotéines/métabolisme , Phosphoprotéines/génétique , Acides picoliniques/pharmacologie , Acides picoliniques/composition chimique , Prolifération cellulaire/effets des médicaments et des substances chimiques , Différenciation cellulaire/effets des médicaments et des substances chimiques , Animaux , Vieillissement de la cellule/effets des médicaments et des substances chimiques , Vieillissement de la cellule/génétique , Protéine-9 associée à CRISPR/métabolisme , Protéine-9 associée à CRISPR/génétique , Régions promotrices (génétique) , Télomère/métabolisme , Télomère/génétique , Protéines proto-oncogènes c-myc/génétique , Protéines proto-oncogènes c-myc/métabolisme , Pyridines/pharmacologie , Pyridines/composition chimique , ADN/métabolisme , ADN/génétique , Souris , Clustered regularly interspaced short palindromic repeats , Cellules HEK293 , Myoblastes/métabolisme , Myoblastes/cytologie , AminoquinoléinesRÉSUMÉ
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.
Sujet(s)
Brevibacillus , Protéine-9 associée à CRISPR , Édition de gène , Brevibacillus/génétique , Brevibacillus/métabolisme , Brevibacillus/enzymologie , Édition de gène/méthodes , Protéine-9 associée à CRISPR/génétique , Protéine-9 associée à CRISPR/métabolisme , Protéine-9 associée à CRISPR/composition chimique , /génétique , /métabolisme , Humains , Systèmes CRISPR-Cas , Ingénierie des protéines/méthodesRÉSUMÉ
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.
Sujet(s)
Édition de gène , Uracil-DNA glycosidase , Uracil-DNA glycosidase/métabolisme , Uracil-DNA glycosidase/génétique , Édition de gène/méthodes , Humains , Ingénierie des protéines/méthodes , ADN/métabolisme , ADN/génétique , Thymine/métabolisme , Deinococcus/génétique , Deinococcus/enzymologie , Deinococcus/métabolisme , Protéine-9 associée à CRISPR/métabolisme , Protéine-9 associée à CRISPR/génétique , Mutation , Cellules HEK293 , Systèmes CRISPR-CasRÉSUMÉ
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.
Sujet(s)
Protéine-9 associée à CRISPR , Systèmes CRISPR-Cas , Cassures double-brin de l'ADN , ADN , Humains , Protéine-9 associée à CRISPR/métabolisme , Protéine-9 associée à CRISPR/génétique , ADN/métabolisme , ADN/génétique , Protéine homologue de MRE11/métabolisme , Protéine homologue de MRE11/génétique , Protéines de liaison à l'ADN/métabolisme , Protéines de liaison à l'ADN/génétique , Protéines associées aux CRISPR/métabolisme , Protéines associées aux CRISPR/génétique , Édition de gène , Endonucleases/métabolisme , Endonucleases/génétique , Protéines bactériennes/métabolisme , Protéines bactériennes/génétique , Endodeoxyribonucleases/métabolisme , Endodeoxyribonucleases/génétique , Réparation de l'ADN par jonction d'extrémités , Clivage de l'ADN , Facteurs de transcription/métabolisme , Facteurs de transcription/génétiqueRÉSUMÉ
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.
Sujet(s)
Protéine-9 associée à CRISPR , , Humains , Protéine-9 associée à CRISPR/génétique , Protéine-9 associée à CRISPR/métabolisme , Protéine-9 associée à CRISPR/composition chimique , /génétique , Streptococcus pyogenes/génétique , Streptococcus pyogenes/enzymologie , Simulation de dynamique moléculaire , Systèmes CRISPR-Cas , Édition de gène/méthodes , Modèles moléculairesRÉSUMÉ
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.
Sujet(s)
Colorimétrie , Or , Techniques d'amplification d'acides nucléiques , Platine , Analyse spectrale Raman , Or/composition chimique , Platine/composition chimique , Salmonella/isolement et purification , Salmonella/génétique , Nanoparticules métalliques/composition chimique , Limite de détection , Benzidines/composition chimique , Protéine-9 associée à CRISPR/métabolisme , Systèmes CRISPR-Cas , Techniques de diagnostic moléculaireRÉSUMÉ
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).
Sujet(s)
Systèmes CRISPR-Cas , Édition de gène , Nanoparticules , Tumeurs , Édition de gène/méthodes , Humains , Nanoparticules/composition chimique , Animaux , Tumeurs/génétique , Tumeurs/thérapie , Épigenèse génétique , Souris , /génétique , Liposomes/composition chimique , Lignée cellulaire tumorale , Lipides/composition chimique , Protéine-9 associée à CRISPR/génétique , Protéine-9 associée à CRISPR/métabolisme , Thérapie génétique/méthodes , Techniques de transfert de gènesRÉSUMÉ
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.
Sujet(s)
Systèmes CRISPR-Cas , Épigenèse génétique , Édition de gène , , Édition de gène/méthodes , Humains , /génétique , Lignée cellulaire , Protéine-9 associée à CRISPR/métabolisme , Protéine-9 associée à CRISPR/génétique , Cellules HEK293RÉSUMÉ
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.
Sujet(s)
Saccharomyces cerevisiae , Saccharomyces cerevisiae/génétique , Humains , Réplication de l'ADN , Protéine-9 associée à CRISPR/génétique , Protéine-9 associée à CRISPR/métabolisme , Duplication de gène , Origine de réplication/génétique , Cassures double-brin de l'ADN , Systèmes CRISPR-Cas/génétiqueRÉSUMÉ
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.
Sujet(s)
Systèmes CRISPR-Cas , Eubacterium , Eubacterium/génétique , Systèmes CRISPR-Cas/génétique , Génie métabolique/méthodes , Recombinaison génétique/génétique , Génome bactérien/génétique , Protéine-9 associée à CRISPR/génétique , Protéine-9 associée à CRISPR/métabolisme , Recombinases/génétique , Recombinases/métabolisme , Génie génétique/méthodes , Édition de gène/méthodes , Famille multigéniqueRÉSUMÉ
The CRISPR/Cas9 system has shown great potential for treating human genetic diseases through gene therapy. However, there are concerns about the safety of this system, specifically related to the use of guide-free Cas9. Previous studies have shown that guide-free Cas9 can induce genomic instability in vitro. However, the in vivo safety risks associated with guide-free Cas9 have not been evaluated, which is necessary for the development of gene therapy in clinical settings. In this study, we used doxycycline-inducible Cas9-expressing pigs to evaluate the safety risks of guide-free Cas9 in vivo. Our findings demonstrated that expression of guide-free Cas9 could induce genomic damages and transcriptome changes in vivo. The severity of the genomic damages and transcriptome changes were correlate with the expression levels of Cas9 protein. Moreover, prolonged expression of Cas9 in pigs led to abnormal phenotypes, including a significant decrease in body weight, which may be attributable to genomic damage-induced nutritional absorption and metabolic dysfunction. Furthermore, we observed an increase in whole-genome and tumor driver gene mutations in pigs with long-term Cas9 expression, raising the risk of tumor occurrence. Our in vivo evaluation of guide-free Cas9 in pigs highlights the necessity of considering and monitoring the detrimental effects of Cas9 alone as genome editing via the CRISPR/Cas9 system is implemented in clinical gene therapy. This research emphasizes the importance of further study and implementation of safety measures to ensure the successful and safe application of the CRISPR/Cas9 system in clinical practice.
Sujet(s)
Protéine-9 associée à CRISPR , Systèmes CRISPR-Cas , Édition de gène , Animaux , Suidae , Systèmes CRISPR-Cas/génétique , Protéine-9 associée à CRISPR/génétique , Protéine-9 associée à CRISPR/métabolisme , /génétique , Humains , Thérapie génétiqueRÉSUMÉ
In their recent structural work, Eggers et al.1 rationalize how key mutations in the WED domain of the compact and thermostable Geobacillus stearothermophilus Cas9 bolster its editing efficiency in mammalian cells, and they use these insights to rationally improve another Cas9.
Sujet(s)
Protéine-9 associée à CRISPR , Édition de gène , Édition de gène/méthodes , Protéine-9 associée à CRISPR/métabolisme , Protéine-9 associée à CRISPR/génétique , Geobacillus stearothermophilus/génétique , Geobacillus stearothermophilus/enzymologie , Systèmes CRISPR-Cas , Humains , Mutation , Protéines bactériennes/métabolisme , Protéines bactériennes/génétique , Protéines bactériennes/composition chimique , AnimauxRÉSUMÉ
DNA base editors enable direct editing of adenine (A), cytosine (C), or guanine (G), but there is no base editor for direct thymine (T) editing currently. Here we develop two deaminase-free glycosylase-based base editors for direct T editing (gTBE) and C editing (gCBE) by fusing Cas9 nickase (nCas9) with engineered human uracil DNA glycosylase (UNG) variants. By several rounds of structure-informed rational mutagenesis on UNG in cultured human cells, we obtain gTBE and gCBE with high activity of T-to-S (i.e., T-to-C or T-to-G) and C-to-G conversions, respectively. Furthermore, we conduct parallel comparison of gTBE/gCBE with those recently developed using other protein engineering strategies, and find gTBE/gCBE show the outperformance. Thus, we provide several base editors, gTBEs and gCBEs, with corresponding engineered UNG variants, broadening the targeting scope of base editors.
Sujet(s)
Protéine-9 associée à CRISPR , Édition de gène , Ingénierie des protéines , Uracil-DNA glycosidase , Humains , Édition de gène/méthodes , Uracil-DNA glycosidase/métabolisme , Uracil-DNA glycosidase/génétique , Ingénierie des protéines/méthodes , Protéine-9 associée à CRISPR/métabolisme , Protéine-9 associée à CRISPR/génétique , Cytosine/métabolisme , Thymine/métabolisme , Systèmes CRISPR-Cas , Cellules HEK293 , Mutagenèse , Guanine/métabolisme , ADN/métabolisme , ADN/génétiqueRÉSUMÉ
CRISPR/Cas systems are perspective molecular tools for targeted manipulation with genetic materials, such as gene editing, regulation of gene transcription, modification of epigenome etc. While CRISPR/Cas systems proved to be highly effective for correcting genetic disorders and treating infectious diseases and cancers in experimental settings, clinical translation of these results is hampered by the lack of efficient CRISPR/Cas delivery vehicles. Modern synthetic nanovehicles based on organic and inorganic polymers have many disadvantages, including toxicity issues, the lack of targeted delivery, and complex and expensive production pipelines. In turn, exosomes are secreted biological nanoparticles that exhibit high biocompatibility, physico-chemical stability, and the ability to cross biological barriers. Early clinical trials found no toxicity associated with exosome injections. In the recent years, exosomes have been considered as perspective delivery vehicles for CRISPR/Cas systems in vivo. The aim of this study was to analyze the efficacy of CRISPR/Cas stochastic packaging into exosomes for several human cell lines. Here, we show that Cas9 protein is effectively localized into the compartment of intracellular exosome biogenesis, but stochastic packaging of Cas9 into exosomes turns to be very low (~1%). As such, stochastic packaging of Cas9 protein is very ineffective and cannot be used for gene editing purposes. Developing novel tools and technologies for loading CRISPR/Cas systems into exosomes is needed.
Sujet(s)
Systèmes CRISPR-Cas , Exosomes , Édition de gène , Exosomes/métabolisme , Exosomes/génétique , Humains , Édition de gène/méthodes , Protéine-9 associée à CRISPR/génétique , Protéine-9 associée à CRISPR/métabolismeRÉSUMÉ
The genome editing approach by clustered regularly interspaced short palindromic repeats (CRISPR)/associated protein 9 (CRISPR/Cas9) is a revolutionary advancement in genetic engineering. Owing to its simple design and powerful genome-editing capability, it offers a promising strategy for the treatment of different infectious, metabolic, and genetic diseases. The crystal structure of Streptococcus pyogenes Cas9 (SpCas9) in complex with sgRNA and its target DNA at 2.5 Å resolution reveals a groove accommodating sgRNA:DNA heteroduplex within a bilobate architecture with target recognition (REC) and nuclease (NUC) domains. The presence of a PAM is significantly required for target recognition, R-loop formation, and strand scission. Recently, the spatiotemporal control of CRISPR/Cas9 genome editing has been considerably improved by genetic, chemical, and physical regulatory strategies. The use of genetic modifiers anti-CRISPR proteins, cell-specific promoters, and histone acetyl transferases has uplifted the application of CRISPR/Cas9 as a future-generation genome editing tool. In addition, interventions by chemical control, small-molecule activators, oligonucleotide conjugates and bioresponsive delivery carriers have improved its application in other areas of biological fields. Furthermore, the intermediation of physical control by using heat-, light-, magnetism-, and ultrasound-responsive elements attached to this molecular tool has revolutionized genome editing further. These strategies significantly reduce CRISPR/Cas9's undesirable off-target effects. However, other undesirable effects still offer some challenges for comprehensive clinical translation using this genome-editing approach. In this review, we summarize recent advances in CRISPR/Cas9 structure, mechanistic action, and the role of small-molecule activators, inhibitors, promoters, and physical approaches. Finally, off-target measurement approaches, challenges, future prospects, and clinical applications are discussed.