RESUMO
Severe congenital neutropenia (CN) is an inherited pre-leukemia bone marrow failure syndrome commonly caused by autosomal-dominant ELANE mutations (ELANE-CN). ELANE-CN patients are treated with daily injections of recombinant human granulocyte colony-stimulating factor (rhG-CSF). However, some patients do not respond to rhG-CSF, and approximately 15% of ELANE-CN patients develop myelodysplasia or acute myeloid leukemia. Here, we report the development of a curative therapy for ELANE-CN through inhibition of ELANE mRNA expression by introducing two single-strand DNA breaks at the opposing DNA strands of the ELANE promoter TATA box using CRISPR-Cas9D10A nickases-termed MILESTONE. This editing effectively restored defective neutrophil differentiation of ELANE-CN CD34+ hematopoietic stem and progenitor cells (HSPCs) in vitro and in vivo, without affecting the functions of the edited neutrophils. CRISPResso analysis of the edited ELANE-CN CD34+ HSPCs revealed on-target efficiencies of over 90%. Simultaneously, GUIDE-seq, CAST-Seq, and rhAmpSeq indicated a safe off-target profile with no off-target sites or chromosomal translocations. Taken together, ex vivo gene editing of ELANE-CN HSPCs using MILESTONE in the setting of autologous stem cell transplantation could be a universal, safe, and efficient gene therapy approach for ELANE-CN patients.
Assuntos
Sistemas CRISPR-Cas , Síndrome Congênita de Insuficiência da Medula Óssea , Edição de Genes , Terapia Genética , Elastase de Leucócito , Neutropenia , Regiões Promotoras Genéticas , Edição de Genes/métodos , Humanos , Neutropenia/congênito , Neutropenia/terapia , Neutropenia/genética , Terapia Genética/métodos , Síndrome Congênita de Insuficiência da Medula Óssea/terapia , Síndrome Congênita de Insuficiência da Medula Óssea/genética , Elastase de Leucócito/genética , Elastase de Leucócito/metabolismo , Animais , Camundongos , Neutrófilos/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Mutação , Modelos Animais de Doenças , Fator Estimulador de Colônias de Granulócitos/genética , Doenças Genéticas Ligadas ao Cromossomo X/terapia , Doenças Genéticas Ligadas ao Cromossomo X/genéticaRESUMO
Cas9 transgenes can be employed for genome editing in mouse zygotes. However, using transgenic instead of exogenous Cas9 to produce gene-edited animals creates unique issues including ill-defined transgene integration sites, the potential for prolonged Cas9 expression in transgenic embryos, and increased genotyping burden. To overcome these issues, we generated mice harboring an oocyte-specific, Gdf9 promoter driven, Cas9 transgene (Gdf9-Cas9) targeted as a single copy into the Hprt1 locus. The X-linked Hprt1 locus was selected because it is a defined integration site that does not influence transgene expression, and breeding of transgenic males generates obligate transgenic females to serve as embryo donors. Using microinjections and electroporation to introduce sgRNAs into zygotes derived from transgenic dams, we demonstrate that Gdf9-Cas9 mediates genome editing as efficiently as exogenous Cas9 at several loci. We show that genome editing efficiency is independent of transgene inheritance, verifying that maternally derived Cas9 facilitates genome editing. We also show that paternal inheritance of Gdf9-Cas9 does not mediate genome editing, confirming that Gdf9-Cas9 is not expressed in embryos. Finally, we demonstrate that off-target mutagenesis is equally rare when using transgenic or exogenous Cas9. Together, these results show that the Gdf9-Cas9 transgene is a viable alternative to exogenous Cas9.
Assuntos
Sistemas CRISPR-Cas , Edição de Genes , Feminino , Masculino , Camundongos , Animais , Edição de Genes/métodos , RNA Guia de Sistemas CRISPR-Cas , Mutação , Zigoto/metabolismo , Animais Geneticamente Modificados , OócitosRESUMO
The Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)/Cas9 system, a groundbreaking innovation in genetic engineering, has revolutionized our approach to surmounting complex diseases, culminating in CASGEVY™ approved for sickle cell anemia. Derived from a microbial immune defense mechanism, CRISPR/Cas9, characterized as precision, maneuverability and universality in gene editing, has been harnessed as a versatile tool for precisely manipulating DNA in mammals. In the process of applying it to practice, the consecutive exploitation of novel orthologs and variants never ceases. It's conducive to understanding the essentialities of diseases, particularly cancer, which is crucial for diagnosis, prevention, and treatment. CRISPR/Cas9 is used not only to investigate tumorous genes functioning but also to model disparate cancers, providing valuable insights into tumor biology, resistance, and immune evasion. Upon cancer therapy, CRISPR/Cas9 is instrumental in developing individual and precise cancer therapies that can selectively activate or deactivate genes within tumor cells, aiming to cripple tumor growth and invasion and sensitize cancer cells to treatments. Furthermore, it facilitates the development of innovative treatments, enhancing the targeting efficiency of reprogrammed immune cells, exemplified by advancements in CAR-T regimen. Beyond therapy, it is a potent tool for screening susceptible genes, offering the possibility of intervening before the tumor initiative or progresses. However, despite its vast potential, the application of CRISPR/Cas9 in cancer research and therapy is accompanied by significant efficacy, efficiency, technical, and safety considerations. Escalating technology innovations are warranted to address these issues. The CRISPR/Cas9 system is revolutionizing cancer research and treatment, opening up new avenues for advancements in our understanding and management of cancers. The integration of this evolving technology into clinical practice promises a new era of precision oncology, with targeted, personalized, and potentially curative therapies for cancer patients.
Assuntos
Sistemas CRISPR-Cas , Neoplasias , Medicina de Precisão , Humanos , Sistemas CRISPR-Cas/genética , Medicina de Precisão/métodos , Neoplasias/genética , Neoplasias/terapia , Edição de Genes/métodos , Animais , Oncologia/métodos , Oncologia/tendênciasRESUMO
Introduction: Breast cancer results from tissue degradation caused by environmental and genetic factors that affect cells in the body. Matrix metalloproteinases, such as MMP-2 and MMP-9, are considered potential putative markers for tumor diagnosis in clinical validation due to their easy detection in body fluids. In addition, recent reports have suggested multiple roles for MMPs, rather than simply degeneration of the extracellular matrix, which comprises mobilizing growth factors and processing surface molecules. Methods: In this study, the chemotherapeutic effects of anthraquinone (AQ) extracted from edible mushrooms (Pleurotus ostreatus Jacq. ex Fr.) cells was examined in MCF-7 breast cancer cells. The cytotoxic potential and oxidative stress induced by purified anthraquinone were assessed in MCF-7 cells using MTT and ROS estimation assays. Gelatin Zymography, and DNA fragmentation assays were performed to examine MMP expression and apoptotic induction in the MCF-7 cells treated with AQ. The genes crucial for mutations were examined, and the mutated RNA knockout plausibility was analyzed using the CRISPR spcas9 genome editing software. Results: MCF-7 cells were attenuated in a concentration-dependent manner by the administration of AQ purified from P. ostreatus compared with the standard anticancer drug paclitaxel. AQ supplementation decreased oxidative stress and mitochondrial impairment in MCF-7 cells. Treatment with AQ and AQ with paclitaxel consistently decreased the expression of crucial marker genes such as MMP2 and MMP9. The mutated genes MMP2, MMP7, and MMP9 were assessed and observed to reveal four putative gene knockdown potentials for breast cancer treatment. Conclusions: The synergistic application of AQ and paclitaxel exerted a strong inhibitory effect on the MCF-7 breast cancer cells. Extensive studies are imperative to better understand the action of bioactive mixes on the edible oyster fungus P. ostreatus. The gene knockout potential detected by CRISPR SpCas9 will aid in elite research into anticancer treatments.
Assuntos
Antraquinonas , Apoptose , Neoplasias da Mama , Metaloproteinase 2 da Matriz , Metaloproteinase 9 da Matriz , Pleurotus , Humanos , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/patologia , Neoplasias da Mama/genética , Antraquinonas/farmacologia , Células MCF-7 , Metaloproteinase 9 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/genética , Feminino , Apoptose/efeitos dos fármacos , Apoptose/genética , Metaloproteinase 2 da Matriz/genética , Metaloproteinase 2 da Matriz/metabolismo , Pleurotus/química , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacosRESUMO
The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9 system is a revolutionary tool for precise genome editing across various cell types. Ribonucleoproteins (RNPs), encompassing the Cas9 protein and guide RNA (gRNA), have emerged as a promising technique due to their increased specificity and reduced off-target effects. This method eliminates the need for plasmid DNA introduction, thereby preventing potential integration of foreign DNA into the target cell genome. Given the requirement for large quantities of highly purified protein in various Cas9 studies, we present an efficient and simple method for the preparation of recombinant Streptococcus pyogenes Cas9 (SpCas9) protein. This method leverages the Small Ubiquitin Like Modifier(SUMO) tag system, which includes metal-affinity chromatography followed by anion-exchange chromatography purification. Furthermore, we compare two methods of CRISPR-Cas9 system delivery into cells: transfection with plasmid DNA encoding the CRISPR-Cas9 system and RNP transfection with the Cas9-gRNA complex. We estimate the efficiency of genomic editing and protein lifespan post-transfection. Intriguingly, we found that RNP treatment of cells, even in the absence of a transfection system, is a relatively efficient method for RNP delivery into cell culture. This discovery is particularly promising as it can significantly reduce cytotoxicity, which is crucial for certain cell cultures such as induced pluripotent stem cells (iPSCs).
Assuntos
Sistemas CRISPR-Cas , RNA Guia de Sistemas CRISPR-Cas , Sistemas CRISPR-Cas/genética , Ribonucleoproteínas/genética , Ribonucleoproteínas/metabolismo , Edição de Genes/métodos , Proteína 9 Associada à CRISPR/genética , Proteína 9 Associada à CRISPR/metabolismo , DNARESUMO
Streptococcus pyogenes Cas9 (SpCas9) is the most popular tool in gene editing; however, off-target mutagenesis is one of the biggest impediments in its application. In our previous study, we proposed the HH theory, which states that sgRNA/DNA hybrid (hybrid) extrusion-induced enhancement of hydrophobic interactions between the hybrid and REC3/HNH is a key factor in cleavage initiation. Based on the HH theory, we analyzed the interactions between the REC3 domain and hybrid and obtained 8 mutant sites. We designed 8 SpCas9 variants (V1-V8), used digital droplet PCR to assess SpCas9-induced DNA indels in human cells, and developed high-fidelity variants. Thus, the HH theory may be employed to further optimize SpCas9-mediated genome editing systems, and the resultant V3, V6, V7, and V8 SpCas9 variants may be valuable for applications requiring high-precision genome editing.
Assuntos
Proteína 9 Associada à CRISPR , Sistemas CRISPR-Cas , Edição de Genes , Streptococcus pyogenes , Humanos , Edição de Genes/métodos , Proteína 9 Associada à CRISPR/genética , Proteína 9 Associada à CRISPR/metabolismo , Streptococcus pyogenes/genética , Streptococcus pyogenes/enzimologia , Células HEK293 , Mutação INDEL , RNA Guia de Sistemas CRISPR-Cas/genética , RNA Guia de Sistemas CRISPR-Cas/metabolismo , DNA/genética , DNA/metabolismo , DNA/químicaRESUMO
CRISPR/Cas systems are some of the most promising tools for therapeutic genome editing. The use of these systems is contingent on the optimal designs of guides and homology-directed repair (HDR) templates. While this design can be achieved in silico, validation and further optimization are usually performed with the help of reporter systems. Here, we describe a novel reporter system, termed BETLE, that allows for the fast, sensitive, and cell-specific detection of genome editing and template-specific HDR by encoding multiple reporter proteins in different open-reading frames. Out-of-frame non-homologous end joining (NHEJ) leads to the expression of either secretable NanoLuc luciferase, enabling a highly sensitive and low-cost analysis of editing, or fluorescent mTagBFP2, allowing for the enumeration and tissue-specific localization of genome-edited cells. BETLE includes a site to validate CRISPR/Cas systems for a sequence-of-interest, making it broadly adaptable. We evaluated BETLE using a defective moxGFP with a 39-base-pair deletion and showed spCas9, saCas9, and asCas12a editing as well as sequence-specific HDR and the repair of moxGFP in cell lines with single and multiple reporter integrants. Taken together, these data show that BETLE allows for the rapid detection and optimization of CRISPR/Cas genome editing and HDR in vitro and represents a state-of the art tool for future applications in vivo.
Assuntos
Sistemas CRISPR-Cas , Quebras de DNA de Cadeia Dupla , Sistemas CRISPR-Cas/genética , Edição de Genes , Reparo do DNA por Junção de Extremidades , GenomaRESUMO
The CRISPR/Cas9 system is a robust, efficient, and cost-effective gene editing tool widely adopted in translational studies of ocular diseases. However, in vivo CRISPR-based editing in animal models poses challenges such as the efficient delivery of the CRISPR components in viral vectors with limited packaging capacity and a Cas9-associated immune response. Using a germline Cas9-expressing mouse model would help to overcome these limitations. Here, we evaluated the long-term effects of SpCas9 expression on retinal morphology and function using Rosa26-Cas9 knock-in mice. We observed abundant SpCas9 expression in the RPE and retina of Rosa26-Cas9 mice using the real-time polymerase chain reaction (RT-PCR), Western blotting, and immunostaining. SD-OCT imaging and histological analysis of the RPE, retinal layers, and vasculature showed no apparent structural abnormalities in adult and aged Cas9 mice. Full-field electroretinogram of adult and aged Cas9 mice showed no long-term functional changes in the retinal tissues because of constitutive Cas9 expression. The current study showed that both the retina and RPE maintain their phenotypic and functional features in Cas9 knock-in mice, establishing this as an ideal animal model for developing therapeutics for retinal diseases.
Assuntos
Sistemas CRISPR-Cas , Retina , Camundongos , Animais , Retina/metabolismo , Edição de Genes/métodos , Eletrorretinografia , Vetores GenéticosRESUMO
The efficiency of the RNA-guided AsCas12a nuclease of Acidaminococcus sp. was compared with SpCas9 from Streptococcus pyogenes, for functional genomics in Schistosoma mansoni. We deployed optimized conditions for the ratio of guide RNAs to the nuclease, donor templates, and electroporation parameters, to target a key schistosome enzyme termed omega-1. Programmed cleavages catalyzed by Cas12a and Cas9 resulted in staggered- and blunt-ended strand breaks, respectively. AsCas12a was more efficient than SpCas9 for gene knockout, as determined by TIDE analysis. CRISPResso2 analysis confirmed that most mutations were deletions. Knockout efficiency of both nucleases markedly increased in the presence of single-stranded oligodeoxynucleotide (ssODN) template. With AsCas12a, ssODNs representative of both the non-CRISPR target (NT) and target (T) strands were tested, resulting in KO efficiencies of 15.67, 28.71, and 21.43% in the SpCas9 plus ssODN, AsCas12a plus NT-ssODN, and AsCas12a plus T-ssODN groups, respectively. Trans-cleavage against the ssODNs by activated AsCas12a was not apparent in vitro. SpCas9 catalyzed more precise transgene insertion, with knock-in efficiencies of 17.07% for the KI_Cas9 group, 14.58% for KI_Cas12a-NT-ssODN, and 12.37% for KI_Cas12a-T-ssODN. Although AsCas12a induced fewer mutations per genome than SpCas9, the phenotypic impact on transcription and expression of omega-1 was similar for both nucleases.
Assuntos
Técnicas de Inativação de Genes , Genes de Protozoários , Loci Gênicos , RNA Guia de Cinetoplastídeos/metabolismo , Reparo de DNA por Recombinação , Ribonucleases/genética , Schistosoma mansoni/genética , Animais , Sequência de Bases , Sistemas CRISPR-Cas/genética , Catálise , Feminino , Dosagem de Genes , Humanos , Mutação/genética , Oligonucleotídeos/metabolismo , Reparo de DNA por Recombinação/genética , Padrões de Referência , Transcrição Gênica , TransgenesRESUMO
The CRISPR/Cas9 system is now commonly employed for genome editing in various plants such as Arabidopsis, rice and tobacco. In general, in genome editing of the Arabidopsis genome, the SpCas9 and guide RNA genes are introduced into the genome by the floral dip method. Mutations induced in the target sequence by SpCas9 are confirmed after selecting transformants by screening the T1 seed population. The advantage of this method is that genome-edited plants can be isolated easily. However, mutation efficiency in Arabidopsis using SpCas9 is not as high as that achieved in rice and tobacco, which are subjected to a tissue culture step. In this study, we compared four promoters and found that the parsley UBIQITIN promoter is highly active in Arabidopsis meristem tissue. Furthermore, we examined whether a simple heat treatment could improve mutation efficiency in Arabidopsis. Just one heat treatment at 37°C for 24 h increased the mutation efficiency at all four target sites from 3 to 42%, 43 to 62%, 54 to 75% and 89 to 91%, without detectable off-target mutations. We recommend heat treatment of plate-grown plants at 37°C for 24 h as a simple method to increase the efficiency of CRISPR/Cas9-mediated mutagenesis in Arabidopsis.
Assuntos
Arabidopsis/genética , Proteína 9 Associada à CRISPR/genética , Sistemas CRISPR-Cas , Edição de Genes/métodos , Genoma de Planta , Temperatura Alta , Arabidopsis/metabolismo , Meristema/metabolismo , Mutação , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , Regiões Promotoras GenéticasRESUMO
The Streptococcus pyogenes Cas9 protein (SpCas9), a component of CRISPR-based immune system in microbes, has become commonly utilized for genome editing. This nuclease forms a ribonucleoprotein (RNP) complex with guide RNA (gRNA) which induces Cas9 structural changes and triggers its cleavage activity. Here, electronic circular dichroism (ECD) spectroscopy was used to confirm the RNP formation and to determine its individual components. The ECD spectra had characteristic features differentiating Cas9 and gRNA, the former showed a negative/positive profile with maxima located at 221, 209 and 196 nm, while the latter revealed positive/negative/positive/negative pattern with bands observed at 266, 242, 222 and 209 nm, respectively. For the first time, the experimental ECD spectrum of the gRNA:Cas9 RNP complex is presented. It exhibits a bisignate positive/negative ECD couplet with maxima at 273 and 235 nm, and it differs significantly from individual spectrum of each RNP components. Additionally, the Cas9 protein and RNP complex retained biological activity after ECD measurements and they were able to bind and cleave DNA in vitro. Hence, we conclude that ECD spectroscopy can be considered as a quick and non-destructive method of monitoring conformational changes of the Cas9 protein as a result of Cas9 and gRNA interaction, and identification of the gRNA:Cas9 RNP complex.
Assuntos
Proteína 9 Associada à CRISPR/química , Sistemas CRISPR-Cas , DNA/química , RNA Guia de Cinetoplastídeos/química , Ribonucleoproteínas/química , Streptococcus pyogenes/química , Pareamento de Bases , Sequência de Bases , Sítios de Ligação , Proteína 9 Associada à CRISPR/genética , Proteína 9 Associada à CRISPR/metabolismo , Dicroísmo Circular , DNA/genética , DNA/metabolismo , Edição de Genes/métodos , Modelos Moleculares , Conformação de Ácido Nucleico , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , RNA Guia de Cinetoplastídeos/genética , RNA Guia de Cinetoplastídeos/metabolismo , Ribonucleoproteínas/genética , Ribonucleoproteínas/metabolismo , Streptococcus pyogenes/enzimologiaRESUMO
Genetic resources for the model plant Arabidopsis comprise mutant lines defective in almost any single gene in reference accession Columbia. However, gene redundancy and/or close linkage often render it extremely laborious or even impossible to isolate a desired line lacking a specific function or set of genes from segregating populations. Therefore, we here evaluated strategies and efficiencies for the inactivation of multiple genes by Cas9-based nucleases and multiplexing. In first attempts, we succeeded in isolating a mutant line carrying a 70 kb deletion, which occurred at a frequency of ~ 1.6% in the T2 generation, through PCR-based screening of numerous individuals. However, we failed to isolate a line lacking Lhcb1 genes, which are present in five copies organized at two loci in the Arabidopsis genome. To improve efficiency of our Cas9-based nuclease system, regulatory sequences controlling Cas9 expression levels and timing were systematically compared. Indeed, use of DD45 and RPS5a promoters improved efficiency of our genome editing system by approximately 25-30-fold in comparison to the previous ubiquitin promoter. Using an optimized genome editing system with RPS5a promoter-driven Cas9, putatively quintuple mutant lines lacking detectable amounts of Lhcb1 protein represented approximately 30% of T1 transformants. These results show how improved genome editing systems facilitate the isolation of complex mutant alleles, previously considered impossible to generate, at high frequency even in a single (T1) generation.
Assuntos
Arabidopsis/genética , Proteína 9 Associada à CRISPR/genética , Edição de Genes/métodos , Alelos , Proteína 9 Associada à CRISPR/metabolismo , Sistemas CRISPR-Cas , Deleção de Genes , Genoma de Planta , Mutação , Regiões Promotoras Genéticas , Ubiquitina/genéticaRESUMO
The primary CO2-fixing enzyme Rubisco limits the productivity of plants. The small subunit of Rubisco (SSU) can influence overall Rubisco levels and catalytic efficiency, and is now receiving increasing attention as a potential engineering target to improve the performance of Rubisco. However, SSUs are encoded by a family of nuclear rbcS genes in plants, which makes them challenging to engineer and study. Here we have used CRISPR/Cas9 [clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated protein 9] and T-DNA insertion lines to generate a suite of single and multiple gene knockout mutants for the four members of the rbcS family in Arabidopsis, including two novel mutants 2b3b and 1a2b3b. 1a2b3b contained very low levels of Rubisco (~3% relative to the wild-type) and is the first example of a mutant with a homogenous Rubisco pool consisting of a single SSU isoform (1B). Growth under near-outdoor levels of light demonstrated Rubisco-limited growth phenotypes for several SSU mutants and the importance of the 1A and 3B isoforms. We also identified 1a1b as a likely lethal mutation, suggesting a key contributory role for the least expressed 1B isoform during early development. The successful use of CRISPR/Cas here suggests that this is a viable approach for exploring the functional roles of SSU isoforms in plants.
Assuntos
Arabidopsis , Ribulose-Bifosfato Carboxilase , Arabidopsis/genética , Arabidopsis/metabolismo , Sistemas CRISPR-Cas , Técnicas de Inativação de Genes , Mutação , Fenótipo , Ribulose-Bifosfato Carboxilase/genética , Ribulose-Bifosfato Carboxilase/metabolismoRESUMO
Cas12e proteins (formerly CasX) form a distinct subtype of Class II type V CRISPR-Cas effectors. Recently, it was shown that DpbCas12e from Deltaproteobacteria and PlmCas12e from Planctomycetes can introduce programmable double-stranded breaks in mammalian genomes. Thus, along with Cas9 and Cas12a Class II effectors, Cas12e could be harnessed for genome editing and engineering. The location of cleavage points in DNA targets is important for application of Cas nucleases in biotechnology. DpbCas12e was reported to produce extensive 5'-overhangs at cleaved targets, which can make it superior for some applications. Here, we used high throughput sequencing to precisely map the DNA cut site positions of DpbCas12e on several DNA targets. In contrast to previous observations, our results demonstrate that DNA cleavage pattern of Cas12e is very similar to that of Cas12a: DpbCas12e predominantly cleaves DNA after nucleotide position 17-19 downstream of PAM in the non-target DNA strand, and after the 22nd position of target strand, producing 3-5 nucleotide-long 5'-overhangs. We also show that reduction of spacer sgRNA sequence from 20nt to 16nt shifts Cas12e cleavage positions on the non-target DNA strand closer to the PAM, producing longer 6-8nt 5'-overhangs. Overall, these findings advance the understanding of Cas12e endonucleases and may be useful for developing of DpbCas12e-based biotechnology instruments.
Assuntos
Proteínas Associadas a CRISPR/metabolismo , Sistemas CRISPR-Cas , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Clivagem do RNA , RNA Guia de Cinetoplastídeos/genética , Sequência de Bases , Sítios de Ligação , Biologia Computacional/métodos , Edição de Genes , Modelos Moleculares , Conformação de Ácido Nucleico , RNA Guia de Cinetoplastídeos/metabolismo , Proteínas RecombinantesRESUMO
Genome editing has become a major tool for both functional studies and plant breeding in several species. Besides generating knockouts through the classical CRISPR-Cas9 system, recent development of CRISPR base editing holds great and exciting opportunities for the production of gain-of-function mutants. The PAM requirement is a strong limitation for CRISPR technologies such as base editing, because the base substitution mainly occurs in a small edition window. As precise single amino-acid substitution can be responsible for functions associated to some domains or agronomic traits, development of Cas9 variants with relaxed PAM recognition is of upmost importance for gene function analysis and plant breeding. Recently, the SpCas9-NG variant that recognizes the NGN PAM has been successfully tested in plants, mainly in monocotyledon species. In this work, we studied the efficiency of SpCas9-NG in the model moss Physcomitrella patens and two Solanaceae crops (Solanum lycopersicum and Solanum tuberosum) for both classical CRISPR-generated gene knock-out and cytosine base editing. We showed that the SpCas9-NG greatly expands the scope of genome editing by allowing the targeting of non-canonical NGT and NGA PAMs. The CRISPR toolbox developed in our study opens up new gene function analysis and plant breeding perspectives for model and crop plants.
Assuntos
Bryopsida/genética , Proteína 9 Associada à CRISPR/genética , Edição de Genes/métodos , Solanum lycopersicum/genética , Solanum tuberosum/genética , Substituição de Aminoácidos/genética , Sistemas CRISPR-Cas/genética , Produtos Agrícolas/genética , Plantas Geneticamente Modificadas/genética , Streptococcus pyogenes/enzimologiaRESUMO
The CRISPR/Cas9 system has greatly improved our ability to engineer targeted mutations in eukaryotic genomes. While CRISPR/Cas9 appears to work universally, the efficiency of targeted mutagenesis and the adverse generation of off-target mutations vary greatly between different organisms. In this study, we report that Arabidopsis plants subjected to heat stress at 37°C show much higher frequencies of CRISPR-induced mutations compared to plants grown continuously at the standard temperature (22°C). Using quantitative assays relying on green fluorescent protein (GFP) reporter genes, we found that targeted mutagenesis by CRISPR/Cas9 in Arabidopsis is increased by approximately 5-fold in somatic tissues and up to 100-fold in the germline upon heat treatment. This effect of temperature on the mutation rate is not limited to Arabidopsis, as we observed a similar increase in targeted mutations by CRISPR/Cas9 in Citrus plants exposed to heat stress at 37°C. In vitro assays demonstrate that Cas9 from Streptococcus pyogenes (SpCas9) is more active in creating double-stranded DNA breaks at 37°C than at 22°C, thus indicating a potential contributing mechanism for the in vivo effect of temperature on CRISPR/Cas9. This study reveals the importance of temperature in modulating SpCas9 activity in eukaryotes, and provides a simple method to increase on-target mutagenesis in plants using CRISPR/Cas9.
Assuntos
Arabidopsis/genética , Sistemas CRISPR-Cas/genética , Genoma de Planta/genética , Streptococcus pyogenes/enzimologia , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Temperatura Alta , Mutagênese Sítio-Dirigida , Mutação , Plantas Geneticamente Modificadas , Estresse FisiológicoRESUMO
Clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9), comprising an RNA-guided DNA endonuclease and a programmable guide RNA (gRNA), is currently recognized to be a powerful genome-editing tool and is widely used in biological science. Despite the usefulness of the system, a protospacer-adjacent motif (PAM) immediately downstream of the target sequence needs to be taken into account in the design of the gRNA, a requirement which limits the flexibility of the CRISPR-based genome-editing system. To overcome this limitation, a Cas9 isolated from Streptococcus pyogenes, namely SpCas9, engineered to develop several variants of Cas9 nuclease, has been generated. SpCas9 recognizes the NGG sequence as the PAM, whereas its variants are capable of interacting with different PAMs. Despite the potential advantage of the Cas9 variants, their functionalities have not previously been tested in the widely used model plant, Arabidopsis thaliana. Here, we developed a plant-specific vector series harboring SpCas9-VQR (NGAN or NGNG) or SpCas9-EQR (NGAG) and evaluated their functionalities. These modified Cas9 nucleases efficiently introduced mutations into the CLV3 and AS1 target genes using gRNAs that were compatible with atypical PAMs. Furthermore, the generated mutations were passed on to their offspring. This study illustrated the usefulness of the SpCas9 variants because the ability to generate heritable mutations will be of great benefit in molecular genetic analyses. A greater number of potential SpCas9-variant-recognition sites in these genes are predicted, compared with those of conventional SpCas9. These results demonstrated the usefulness of the SpCas9 variants for genome editing in the field of plant science research.
Assuntos
Arabidopsis/genética , Proteína 9 Associada à CRISPR/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Edição de Genes , RNA Guia de Cinetoplastídeos/genética , Streptococcus pyogenes/enzimologia , Proteína 9 Associada à CRISPR/metabolismo , Sistemas CRISPR-Cas , Mutagênese , Mutação , Especificidade da Espécie , Streptococcus pyogenes/genéticaRESUMO
Myotonic dystrophy type 1 (DM1) is caused by a CTG nucleotide repeat expansion within the 3' UTR of the Dystrophia Myotonica protein kinase gene. In this study, we explored therapeutic genome editing using CRISPR/Cas9 via targeted deletion of expanded CTG repeats and targeted insertion of polyadenylation signals in the 3' UTR upstream of the CTG repeats to eliminate toxic RNA CUG repeats. We found paired SpCas9 or SaCas9 guide RNA induced deletion of expanded CTG repeats. However, this approach incurred frequent inversion in both the mutant and normal alleles. In contrast, the insertion of polyadenylation signals in the 3' UTR upstream of the CTG repeats eliminated toxic RNA CUG repeats, which led to phenotype reversal in differentiated neural stem cells, forebrain neurons, cardiomyocytes, and skeletal muscle myofibers. We concluded that targeted insertion of polyadenylation signals in the 3' UTR is a viable approach to develop therapeutic genome editing for DM1.
Assuntos
Distrofia Miotônica/genética , Miotonina Proteína Quinase/genética , Células-Tronco Neurais/fisiologia , Expansão das Repetições de Trinucleotídeos/genética , Regiões 3' não Traduzidas , Sistemas CRISPR-Cas/genética , Diferenciação Celular/genética , Edição de Genes/métodos , Terapia Genética/métodos , Células HEK293 , Humanos , Músculo Esquelético/crescimento & desenvolvimento , Miócitos Cardíacos/fisiologia , Distrofia Miotônica/patologia , Distrofia Miotônica/terapia , Neurônios/fisiologia , Sinais de Poliadenilação na Ponta 3' do RNA/genética , RNA Guia de Cinetoplastídeos , TransfecçãoRESUMO
Despite the great achievements in genome editing, accurately detecting mutations induced by sequence-specific nucleases is still a challenge in plants, especially in polyploidy plants. An efficient detection method is particularly vital when the mutation frequency is low or when a large population needs to be screened. Here, we applied purified CRISPR ribonucleoprotein complexes to cleave PCR products for genome-edited mutation detection in hexaploid wheat and diploid rice. We show that this mutation detection method is more sensitive than Sanger sequencing and more applicable than PCR/RE method without the requirement for restriction enzyme site. We also demonstrate that this detection method is especially useful for genome editing in wheat, because target sites are often surrounded by single nucleotide polymorphisms. Using this screening method, we were also able to detect foreign DNA-free tagw2 mutations induced by purified TALEN protein. Finally, we show that partial base editing mutations can also be detected using high-fidelity SpCas9 variants or FnCpf1. The PCR/RNP method is low-cost and widely applicable for rapid detection of genome-edited mutation in plants.