RESUMEN
Versatile genome editing can be facilitated by the insertion of DNA sequences into specific locations. Current protocols involving CRISPR and Cas proteins rely on low efficiency homology-directed repair or non-homologous end joining with modified double-stranded DNA oligonucleotides as donors. Our simple protocol eliminates the need for expensive equipment, chemical and enzymatic donor DNA modification, or plasmid construction by using polyethylene glycol-calcium to deliver non-modified single-stranded DNA oligonucleotides and CRISPR-Cas9 ribonucleoprotein into protoplasts. Plants regenerated via edited protoplasts achieved targeted insertion frequencies of up to 50% in Nicotiana benthamiana and 13.6% in rapid cycling Brassica oleracea without antibiotic selection. Using a 60 nt donor containing 27 nt in each homologous arm, 6/22 regenerated N. benthamiana plants showed targeted insertions, and one contained a precise insertion of a 6 bp HindIII site. The inserted sequences were transmitted to the next generation and invite the possibility of future exploration of versatile genome editing by targeted DNA insertion in plants.
Asunto(s)
Marcación de Gen/métodos , Genoma de Planta , Mutagénesis Insercional , Costos y Análisis de Costo , Edición Génica/economía , Edición Génica/métodos , Marcación de Gen/economía , Protoplastos/citología , Protoplastos/metabolismo , Nicotiana/genéticaRESUMEN
The Cre/LoxP-based conditional knockout technology is a powerful tool for gene function analysis that allows region- and time-specific gene manipulation. However, inserting a pair of LoxP cassettes to generate conditional knockout can be technically challenging and thus time- and resource-consuming. This study proposes an efficient, low-cost method to generate floxed mice using in vitro fertilization and the CRISPR-Cas9 system over two consecutive generations. This method allowed us to produce floxed mice targeting exons 5 and 6 of CaMK1 in a short period of 125 days, using only 16 mice. In addition, we directly edited the genome of fertilized eggs of mice with our target genetic background, C57BL/6 N, to eliminate additional backcrossing steps. We confirmed that the genome of the generated floxed mice was responsive to the Cre protein. This low-cost, time-saving method for generating conditional knockout will facilitate comprehensive, tissue-specific genome analyses.
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Sistemas CRISPR-Cas , Electroporación/métodos , Edición Génica/métodos , Marcación de Gen/métodos , Ratones Noqueados , Neurociencias/métodos , Animales , Secuencia de Bases , Proteína Quinasa Tipo 1 Dependiente de Calcio Calmodulina/genética , Transferencia de Embrión , Exones/genética , Edición Génica/economía , Marcación de Gen/economía , Integrasas , Ratones , Ratones Endogámicos C57BL , Neurociencias/economía , TransgenesRESUMEN
Targeted gene disruption has rapidly become the tool of choice for the analysis of gene and protein function in routinely cultured mammalian cells. Three main technologies capable of irreversibly disrupting gene-expression exist: zinc-finger nucleases, transcription activator-like effector nucleases (TALENs), and the CRISPR/Cas9 system. The desired outcome of the use of any of these technologies is targeted insertions and/or deletions (indels) that result in either a nonsense frame shift or splicing error that disrupts protein expression. Many excellent do-it-yourself systems for TALEN construct assembly are now available at low or no cost to academic researchers. However, for new users, screening for successful gene disruption is still a hurdle. Here, we describe efficient and cost-effective strategies for the generation of gene-disrupted cell lines. Although the focus of this chapter is on the use of TALENs, these strategies can be applied to the use of all three technologies.
Asunto(s)
Marcación de Gen/métodos , Ingeniería Genética/métodos , Nucleasas de los Efectores Tipo Activadores de la Transcripción/genética , Animales , Células Cultivadas , Marcación de Gen/economía , Ingeniería Genética/economía , Humanos , Ratones , Canal Aniónico 2 Dependiente del Voltaje/antagonistas & inhibidores , Canal Aniónico 2 Dependiente del Voltaje/genéticaRESUMEN
RATIONALE: Thousands of mutations across >50 genes have been implicated in inherited cardiomyopathies. However, options for sequencing this rapidly evolving gene set are limited because many sequencing services and off-the-shelf kits suffer from slow turnaround, inefficient capture of genomic DNA, and high cost. Furthermore, customization of these assays to cover emerging targets that suit individual needs is often expensive and time consuming. OBJECTIVE: We sought to develop a custom high throughput, clinical-grade next-generation sequencing assay for detecting cardiac disease gene mutations with improved accuracy, flexibility, turnaround, and cost. METHODS AND RESULTS: We used double-stranded probes (complementary long padlock probes), an inexpensive and customizable capture technology, to efficiently capture and amplify the entire coding region and flanking intronic and regulatory sequences of 88 genes and 40 microRNAs associated with inherited cardiomyopathies, congenital heart disease, and cardiac development. Multiplexing 11 samples per sequencing run resulted in a mean base pair coverage of 420, of which 97% had >20× coverage and >99% were concordant with known heterozygous single nucleotide polymorphisms. The assay correctly detected germline variants in 24 individuals and revealed several polymorphic regions in miR-499. Total run time was 3 days at an approximate cost of $100 per sample. CONCLUSIONS: Accurate, high-throughput detection of mutations across numerous cardiac genes is achievable with complementary long padlock probe technology. Moreover, this format allows facile insertion of additional probes as more cardiomyopathy and congenital heart disease genes are discovered, giving researchers a powerful new tool for DNA mutation detection and discovery.
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Análisis Costo-Beneficio , Cardiopatías/economía , Cardiopatías/genética , Secuenciación de Nucleótidos de Alto Rendimiento/economía , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Secuencia de Bases , Marcación de Gen/economía , Marcación de Gen/métodos , Cardiopatías/diagnóstico , Humanos , Datos de Secuencia Molecular , Polimorfismo de Nucleótido Simple/genética , Factores de TiempoRESUMEN
Recent genome-wide studies have revealed that the majority of the mouse genome is transcribed as non-coding RNAs (ncRNAs) and growing evidence supports the importance of ncRNAs in regulating gene expression and epigenetic processes. However, the low efficiency of conventional gene targeting strategies has hindered the functional study of ncRNAs in vivo, particularly in generating large fragment deletions of long non-coding RNAs (lncRNAs) with multiple expression variants. The bacterial clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) system has recently been applied as an efficient tool for engineering site-specific mutations of protein-coding genes in the genome. In this study, we explored the potential of using the CRISPR/Cas9 system to generate large genomic deletions of lncRNAs in mice. We developed an efficient one-step strategy to target the maternally expressed lncRNA, Rian, on chromosome 12 in mice. We showed that paired sgRNAs can precisely generate large deletions up to 23kb and the deletion efficiency can be further improved up to 33% by combining multiple sgRNAs. The deletion successfully abolished the expression of Rian from the maternally inherited allele, validating the biological relevance of the mutations in studying an imprinted locus. Mutation of Rian has differential effects on expression of nearby genes in different somatic tissues. Taken together, we have established a robust one-step method to engineer large deletions to knockout lncRNA genes with the CRISPR/Cas9 system. Our work will facilitate future functional studies of other lncRNAs in vivo.
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Sistemas CRISPR-Cas , Eliminación de Gen , Marcación de Gen/métodos , Proteínas Nucleares/metabolismo , Animales , Cromosomas de los Mamíferos/genética , Marcación de Gen/economía , Impresión Genómica , Ratones , Proteínas Nucleares/genéticaRESUMEN
The genetic, biochemical and molecular bases of human cardiac disease have been the focus of extensive research efforts for many years. Early animal models of cardiovascular disease used pharmacologic or surgical interventions, or took advantage of naturally occurring genetic abnormalities and the data obtained were largely correlative. The inability to directly alter an organism's genetic makeup and cellular protein content and accurately measure the results of that manipulation precluded rigorous examination of true cause-effect and structure-function relationships. Directed genetic manipulation in the mouse gave researchers the ability to modify and control the mammalian heart's protein content, resulting in the rational design of models that could provide critical links between the mutated or absent protein and disease. Two techniques that have proven particularly useful are transgenesis, which involves the random insertion of ectopic genetic material of interest into a "host" genome, and gene targeting, which utilizes homologous recombination at a pre-selected locus. Initially, transgenesis and gene targeting were used to examine systemic loss-of-function and gain-of-function, respectively, but further refinements in both techniques have allowed for investigations of organ-specific, cell type-specific, developmental stage-sensitive and dose-dependent effects. Genetically engineered animal models of pediatric and adult cardiac disease have proven that, when used appropriately, these tools have the power to extend mere observation to the establishment of true causative proof. We illustrate the power of the general approach by showing how genetically engineered mouse models can define the precise signaling pathways that are affected by the gain-of-function mutation that underlies Noonan syndrome. Increasingly precise and modifiable animal models of human cardiac disease will allow researchers to determine not only pathogenesis, but also guide treatment and the development of novel therapies.
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Modelos Animales de Enfermedad , Marcación de Gen/tendencias , Técnicas de Transferencia de Gen/tendencias , Cardiopatías/diagnóstico , Cardiopatías/genética , Modelos Cardiovasculares , Mutagénesis Insercional/métodos , Animales , Marcación de Gen/economía , Marcación de Gen/métodos , Técnicas de Transferencia de Gen/economía , Corazón/fisiopatología , Cardiopatías/patología , Cardiopatías/fisiopatología , Humanos , Mutagénesis Insercional/economía , SíndromeRESUMEN
Targeted biotech drugs that attack specific biological molecules that cause disease are bringing new benefits even as they foment pricing dynamics that are very different from those of traditional drugs. Targeted drugs tend not to compete with each other even when treating closely related diseases, which makes them resistant to price controls. We can expect the supply of expensive new so-called biotech drugs to continue. But the same properties that generate premium prices also facilitate inventing around successful drugs, eventually leading to vigorous competition despite the lack of generic alternatives.
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Biotecnología/economía , Sistemas de Liberación de Medicamentos/economía , Industria Farmacéutica/economía , Costos de los Medicamentos , Marcación de Gen/economía , Humanos , Estados UnidosRESUMEN
RNA interference, the inhibition of gene expression by double-stranded RNA, provides a powerful tool for functional studies once the sequence of a gene is known. In most mammalian cells, only short molecules can be used because long ones induce the interferon pathway. With the identification of a proper target sequence, the penetration of the oligonucleotides constitutes the most serious limitation in the application of this technique. Here we show that a small interfering RNA (siRNA) targeting the mRNA of the kinesin Eg5 induces a rapid mitotic arrest and provides a convenient assay for the optimization of siRNA transfection. Thus, dose responses can be established for different transfection techniques, highlighting the great differences in response to transfection techniques of various cell types. We report that the calcium phosphate precipitation technique can be an efficient and cost-effective alternative to Oligofectamine in some adherent cells, while electroporation can be efficient for some cells growing in suspension such as hematopoietic cells and some adherent cells. Significantly, the optimal parameters for the electroporation of siRNA differ from those for plasmids, allowing the use of milder conditions that induce less cell toxicity. In summary, a single siRNA leading to an easily assayed phenotype can be used to monitor the transfection of siRNA into any type of proliferating cells of both human and murine origin.
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Marcación de Gen/métodos , Cinesinas/genética , Interferencia de ARN , ARN Interferente Pequeño/farmacología , Transfección/métodos , Proteínas de Xenopus/genética , Fosfatos de Calcio , Adhesión Celular , Permeabilidad de la Membrana Celular , Precipitación Química , Análisis Costo-Beneficio , Portadores de Fármacos , Electroporación , Marcación de Gen/economía , Células HeLa , Humanos , Células K562 , Leucemia Megacarioblástica Aguda/patología , Mitosis/efectos de los fármacos , ARN Mensajero/antagonistas & inhibidores , ARN Interferente Pequeño/genética , Transfección/economía , Células Tumorales CultivadasRESUMEN
We introduce a PCR-based procedure for generating a gene disruption construct. This method depends on DNA fragment fusion by the PCR technique and requires only two steps of PCR to obtain a sufficient amount of the gene disruption construct for one transformation experiment. The first step involves three separate PCR syntheses of a selectable marker cassette and the 5'- and 3'-regions of a target gene. Of the four primers used in amplification of the 5'- and 3'-regions of the target gene, two primers placed proximal to the site of the marker cassette are designed to have sequence tags complementary to the 5'- or 3'-side of the marker cassette. The two primers used in PCR synthesis of the marker cassette are complementary to the tagged primers. By fusion PCR, the 5' and 3' PCR products are linked to the marker cassette via the regions of tagged primers that overlap. A sufficient amount of the disruption construct can be directly amplified with the outermost primers. This method is simple, rapid and relatively inexpensive. In addition, there is the freedom of attaching long flanking regions to any selectable marker cassette.