RESUMEN
CRISPR-Cas12a (Cpf1) is widely used for pathogen detection. However, most Cas12a nucleic acid detection methods are limited by a PAM sequence requirement. Moreover, preamplification and Cas12a cleavage are separate. Here, we developed a one-step RPA-CRISPR detection (ORCD) system unrestricted by the PAM sequence with high sensitivity and specificity that offers one-tube, rapid, and visually observable detection of nucleic acids. In this system, Cas12a detection and RPA amplification are performed simultaneously, without separate preamplification and product transfer steps, and 0.2 copies/µL of DNA and 0.4 copies/µL of RNA can be detected. In the ORCD system, the activity of Cas12a is the key to the nucleic acid detection; specifically, reducing Cas12a activity increases the sensitivity of ORCD assay detection of the PAM target. Furthermore, by combining this detection technique with a nucleic acid extraction-free method, our ORCD system can be used to extract, amplify and detect samples within 30 min, as verified with tests of 82 Bordetella pertussis clinical samples with a sensitivity and specificity of 97.30% and 100% compared with PCR. We also tested 13 SARS-CoV-2 samples with RT-ORCD, and the results were consistent with RT-PCR.
Asunto(s)
COVID-19 , Ácidos Nucleicos , Humanos , SARS-CoV-2 , ARN , Bioensayo , Técnicas de Amplificación de Ácido NucleicoRESUMEN
The vaccinia virus (VACV) was previously used as a vaccine for smallpox eradication. Nowadays, recombinant VACVs are developed as vaccine platforms for infectious disease prevention and cancer treatment. The conventional method for genome editing of the VACV is based on homologous recombination, which is poorly efficient. Recently, the use of CRISPR/Cas9 technology was shown to greatly improve the speed and efficiency of the production of recombinant VACV expressing a heterologous gene. However, the ability to rapidly recover viruses bearing single nucleotide substitutions is still challenging. Notwithstanding, ongoing studies on the VACV and its interaction with the host cell could benefit from viral gene targeted mutagenesis. Here, we present a modified version of the CRISPR/Cas9 system for the rapid selection of mutant VACV carrying point mutations. For this purpose, we introduced a silent mutation into the donor gene (which will replace the wildtype gene) that serves a double function: it is located in the PAM (NGG) sequence, which is essential for Cas9 cleavage, and it alters a restriction site. This silent mutation, once introduced into the VACV genome, allows for rapid selection and screening of mutant viruses carrying a mutation of interest in the targeted gene. As a proof of concept, we produced several recombinant VACVs, with mutations in the E9L gene, upon which, phenotypic analysis was performed.
Asunto(s)
Sistemas CRISPR-Cas , Virus Vaccinia , Secuencia de Bases , Edición Génica/métodos , Mutación Puntual , Virus Vaccinia/genéticaRESUMEN
The realization of the full potential of human pluripotent stem cells (hPSCs), including human induced PSCs (iPSC), relies on the ability to precisely edit their genome in a locus-specific and multiplex manner. Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) serve as a guide for the endonuclease Cas9 (CRISPR-associated protein 9) to recognize and cleave specific strands of DNA that are complementary to the CRISPR sequence. CRISPR/Cas9-mediated editing has become the gold standard for precise genome manipulation as it offers a unique, versatile, and limitless tool for fast, robust, and efficient genome editing. Here, we provide a protocol to successfully generate gene knockout and/or knockin iPSCs. We include detailed information on the design of guide RNAs (gRNAs), T7 endonuclease assay to detect on-target CRISPR/Cas9 editing events, DNA electroporation of the iPSCs with a ribonucleoprotein complex, and single-cell cloning steps for the selection of the genome-edited iPSC clones.
Asunto(s)
Sistemas CRISPR-Cas , Células Madre Pluripotentes Inducidas , Sistemas CRISPR-Cas/genética , ADN/metabolismo , Endonucleasas/genética , Endonucleasas/metabolismo , Técnicas de Inactivación de Genes , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , ARN Guía de Kinetoplastida/genética , ARN Guía de Kinetoplastida/metabolismoRESUMEN
CRISPR/Cas9, once discovered as an adaptive immune system in bacteria, has emerged as a disruptive technology in the field of genetic engineering. Technological advancements in the recent past has enhanced the applicability of CRISPR/Cas9 tool for gene editing, gene therapies, developmental studies and mutational analysis in various model organisms. Zebrafish, one of the excellent animal models, is preferred for conducting CRISPR/Cas9 studies to assess the functional implication of specific genes of interest. CRISPR/Cas9 mediated gene editing techniques, such as, knock-out and knock-in approaches, provide evidences to identify the role of different genes through loss-of-function studies. Also, CRISPR/Cas9 has been proved to be an efficient tool for designing disease models for gene expression studies based on phenotypic screening. The present chapter provides an overview of CRISPR/Cas9 mechanism, different strategies for DNA modifications and gene function analysis, highlighting the translational applications for future prospects, such as screening of drug toxicity and efficacy.
Asunto(s)
Edición Génica , Pez Cebra , Animales , Sistemas CRISPR-Cas/genética , Ingeniería Genética , Terapia Genética , Pez Cebra/genéticaRESUMEN
The variations of microRNA (miRNA) expression can be valuable biomarkers in disease diagnosis and prognosis. However, current miRNA detection techniques mainly rely on reverse transcription and template replication, which suffer from slowness, contamination risk, and sample loss. To address these limitations, here we introduce a cascade toehold-mediated strand displacement reaction (CTSDR) and CRISPR/Cas12a trans-cleavage for highly sensitive fluorescent miRNA sensing, namely CTSDR-Cas12a. In this work, the target miRNA hybridizes with the terminal toehold site of a rationally designed probe and subsequently initiates dynamic CTSDR, leading to enzyme-free target recycling and the production of multiple programmable DNA duplexes. The obtained DNA duplex acts as an activator to trigger Cas12a trans-cleavage, generating significantly amplified fluorescence readout for highly sensitive detection of the miRNA target. Under the optimal conditions, the developed sensing method can detect target miRNA down to 70.28 fM with a wide linear range from 100 fM to 100 pM. In particular, by designing a set of probes and crRNAs, we demonstrate its broad applicability for the detection of six kinds of miRNAs with high sequence specificity. Furthermore, the method can be satisfactorily applied to monitor miR-21 in total RNA extracted from cells and clinical serum samples. Considering the high sensitivity, specificity, universality, and ease of handling, this strategy provides a great potential platform for the detection of miRNA biomarkers in molecular diagnostic practice.
Asunto(s)
Proteínas Bacterianas/genética , Proteínas Asociadas a CRISPR/genética , Sistemas CRISPR-Cas , ADN/genética , Endodesoxirribonucleasas/genética , Edición Génica/métodos , MicroARNs/genética , Nanoestructuras , Técnicas de Amplificación de Ácido Nucleico/métodos , Técnicas Biosensibles/métodos , Donantes de Sangre , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/genética , ADN/metabolismo , Fluorescencia , Células HeLa , Humanos , Células MCF-7 , MicroARNs/metabolismo , Hibridación de Ácido Nucleico/métodos , Sensibilidad y EspecificidadRESUMEN
The 2020 Nobel Prize in Chemistry was awarded to the American scientist Jennifer A. Doudna and the French scientist Emmanuelle Charpentier, in recognition of their discovery in one of the greatest weapons in genetic technology: CRISPR-Cas9 gene scissors. The CRISPR-Cas system is a bacterial defense immune system against exogenous genetic material. Because the system can specifically recognize and cut DNA, this technology is widely used for precise editing of animal, plant, and microbial DNA. The discovery of CRISPR-Cas9 gene scissors enables the tedious and complicated cell gene editing work to be completed in a few weeks or even less, which has promoted the development of gene editing technology in various fields and brought revolutionary influence to the field of life sciences. At the same time, CRISPR gene editing technology has become one of the new therapies for tumors because of its large number of targets and relatively simple operation, and it also makes gene therapy possible. Although the technology still needs to solve technical problems such as off-target and promoter inefficiency, the CRISPR-Cas system will show its unique advantages in more fields with the continuous development of life science and basic medicine.
Asunto(s)
Sistemas CRISPR-Cas , Neoplasias , Animales , Bacterias/genética , ADN , Edición Génica , Neoplasias/genéticaRESUMEN
CRISPR interference (CRISPRi) has been developed as a transcriptional control tool by inactivating the DNA cleavage ability of Cas9 nucleases to produce dCas9 (deactivated Cas9), and leaving dCas9 the ability to specifically bind to the target DNA sequence. CRISPR/Cas9 technology has limitations in designing target-specific single-guide RNA (sgRNA) due to the dependence of protospacer adjacent motif (PAM) (5'-NGG) for binding target DNAs. Reportedly, Cas9-NG recognizing 5'-NG as the PAM sequence has been constructed by removing the dependence on the last base G of PAM through protein engineering of Cas9. In this study, a dCas9-NG protein was engineered by introducing two active site mutations in Cas9-NG, and its ability to regulate transcription was evaluated in the gal promoter in E. coli. Analysis of cell growth rate, D-galactose consumption rate, and gal transcripts confirmed that dCas9-NG can completely repress the promoter by recognizing DNA targets with PAM of 5'-NGG, NGA, NGC, NGT, and NAG. Our study showed possible PAM sequences for dCas9-NG and provided information on target-specific sgRNA design for regulation of both gene expression and cellular metabolism.