Climate change and the diversity of consumer needs require innovative methods to continuously and rapidly modify existing crops for the development of new varieties.In the past decade genome editing ...by CRISPR/Cas and derivatives has emerged as a novel and effective technology for functional studies and gene discovery as well as for breeding new traits and genotypes.The development of novel CRISPR/Cas platforms, methods for the delivery of editing reagents, and methods for controlling gene regulation and detection of mutants have all expanded the scope of genome editing and other CRISPR/Cas-based approaches.
The discovery of the CRISPR/Cas genome-editing system has revolutionized our understanding of the plant genome. CRISPR/Cas has been used for over a decade to modify plant genomes for the study of specific genes and biosynthetic pathways as well as to speed up breeding in many plant species, including both model and non-model crops. Although the CRISPR/Cas system is very efficient for genome editing, many bottlenecks and challenges slow down further improvement and applications. In this review we discuss the challenges that can occur during tissue culture, transformation, regeneration, and mutant detection. We also review the opportunities provided by new CRISPR platforms and specific applications related to gene regulation, abiotic and biotic stress response improvement, and de novo domestication of plants.
The conventional detection model of passive adaptation to pathogen mutations, i.e., developing assays using corresponding antibodies or nucleic acid probes, is difficult to address frequent outbreaks ...of emerging infectious diseases. In particular, adaptive mutations observed in coronaviruses, which increase the affinity of the spike protein with the human cellular receptor hACE2, play pivotal roles in the transmission and immune evasion of coronaviruses. Herein, we developed a multifunctional optical fiber evanescent wave biosensor for the universal assay of coronavirus and affinity analysis of the spike protein interacting with hACE2, namely, My-SPACE. By competitively binding with Cy5.5-hACE2 between coronavirus spike proteins in mobile buffer and that modified on optical fibers from the SARS-CoV-2 wild type, My-SPACE could automatically detect SARS-CoV-2 and its variants within 10 min. My-SPACE demonstrated greater sensitivity and faster results than ELISA for SARS-CoV-2 variants, achieving 100% specificity and 94.10% sensitivity in detecting the Omicron variant in 18 clinical samples. Moreover, the interaction between hACE2 and the coronavirus spike protein was accurately characterized across SARS-CoV-2 mutants, SARS-CoV and hCoV-NL63. The accuracy of the affinity determined by My-SPACE was verified by SPR. This approach enables preliminary assessment of the transmissibility and hazards of emerging coronaviruses. The sensor fibers of My-SPACE can be reused more than 40 times, and the device is compact and easy to use; moreover, it is available as a rapid and cost-effective on-site detection tool adapted to coronavirus variability and as an effective assessment platform for early warning of coronavirus transmission risk.
Onco-proteogenomics aims to understand how changes in a cancer's genome influences its proteome. One challenge in integrating these molecular data is the identification of aberrant protein products ...from mass-spectrometry (MS) datasets, as traditional proteomic analyses only identify proteins from a reference sequence database.
We established proteomic workflows to detect peptide variants within MS datasets. We used a combination of publicly available population variants (dbSNP and UniProt) and somatic variations in cancer (COSMIC) along with sample-specific genomic and transcriptomic data to examine proteome variation within and across 59 cancer cell-lines.
We developed a set of recommendations for the detection of variants using three search algorithms, a split target-decoy approach for FDR estimation, and multiple post-search filters. We examined 7.3 million unique variant tryptic peptides not found within any reference proteome and identified 4771 mutations corresponding to somatic and germline deviations from reference proteomes in 2200 genes among the NCI60 cell-line proteomes.
We discuss in detail the technical and computational challenges in identifying variant peptides by MS and show that uncovering these variants allows the identification of druggable mutations within important cancer genes.
Context: The equivalent mutant problem is a well-known impediment to the adoption of mutation testing in practice. In consequence of its undecidable nature, a complete automated solution is ...unattainable. To worsen the situation, the manual analysis of the generated mutants of a program under test is prohibitive due to their vast number and the complexity of determining their equivalence.
Objective: This paper focuses on the automated identification of equivalent and partially equivalent mutants, i.e. mutants that are equivalent to the original program for a specific subset of paths. To this end, the utilisation of a series of previously proposed data flow patterns is investigated. This study also examines the cross-language nature of these patterns and the killability of the detected partially equivalent mutants.
Method: A tool, named MEDIC (Mutants’ Equivalence DIsCovery), incorporating the aforementioned patterns was developed. Its efficiency and effectiveness were evaluated based on a set of manually analysed mutants from real-world programs, written in the Java programming language. Furthermore, MEDIC was employed to test subjects written in the JavaScript programming language.
Results: MEDIC managed to detect 56% of the examined equivalent mutants in 125 s, providing strong evidence regarding both its effectiveness and efficiency. Additionally, MEDIC was able to identify equivalent mutants in the JavaScript test subjects, lending colour to the cross-language nature of the implemented patterns. Finally, the identified partially equivalent mutant set consisted largely of killable mutants, 16% of which were stubborn ones.
Conclusion: It can be concluded that pattern-based equivalent mutant identification forms a viable approach for combating the equivalent mutant problem. MEDIC automatically detected a considerable number of the manually identified equivalent mutants and was successfully applied to test subjects in all examined programming languages.
A set of universal guidelines is needed to determine the limit of detection (LOD) in PCR‐based analyses of low‐concentration DNA. In particular, environmental DNA (eDNA) studies require sensitive and ...reliable methods to detect rare and cryptic species through shed genetic material in environmental samples. Current strategies for assessing detection limits of eDNA are either too stringent or subjective, possibly resulting in biased estimates of species’ presence. Here, a conservative LOD analysis grounded in analytical chemistry is proposed to correct for overestimated DNA concentrations predominantly caused by the concentration plateau, a nonlinear relationship between expected and measured DNA concentrations. We have used statistical criteria to establish formal mathematical models for both quantitative and droplet digital PCR. To assess the method, a new Grass Carp (Ctenopharyngodon idella) TaqMan assay was developed and tested on both PCR platforms using eDNA in water samples. The LOD adjustment reduced Grass Carp occupancy and detection estimates while increasing uncertainty—indicating that caution needs to be applied to eDNA data without LOD correction. Compared to quantitative PCR, digital PCR had higher occurrence estimates due to increased sensitivity and dilution of inhibitors at low concentrations. Without accurate LOD correction, species occurrence and detection probabilities based on eDNA estimates are prone to a source of bias that cannot be reduced by an increase in sample size or PCR replicates. Other applications also could benefit from a standardized LOD such as GMO food analysis and forensic and clinical diagnostics.
At present, with the accelerated development of the global biotechnology industry, novel transgenic technologies represented by gene editing are developing rapidly. A large number of gene-edited ...products featuring one or a few base indels have been commercialized. These have led to great challenges in the use of traditional nucleic acid detection technology and in safety regulation for genetically modified organisms (GMOs). In this study, we developed a portable clustered regularly interspaced short palindromic repeats/CRISPR-associated proteins 12a-based (CRISPR/Cas12a-based) biosensing platform named Cas12aFVD (fast visual detection) that can be coupled with recombinase polymerase amplification (RPA) for on-site detection of mutants in gene-edited rice in one tube. The detection procedure can be accomplished in 40 min with a visible result, which can be observed by the naked eye under blue light (470–490 nm). By accurate recognition of targets based on Cas12a/CRISPR RNA (crRNA), Cas12aFVD exhibits excellent performance for the detection of two- and three-base deletions, one-base substitution, and one-base insertion mutants with a limit of detection (LOD) of 12 copies/μl showing great potential for mutant detection, especially single-base mutants. The Cas12aFVD biosensing platform is independent of laboratory conditions, making it a promising and pioneering platform for the detection of gene-edited products.
When method-dependent categorical endpoints are available, namely either BPs or ECVs, MICs could aid in selecting the best treatment agent(s). BPs can categorize an isolate as either susceptible or ...resistant while the ECVs/ECOFFs can distinguish the wild type (WT, no known resistance mechanisms) from the Non-WT (NWT, harboring resistant mechanisms). Our literature review focused on the
species complex (SC) and the available methods and categorization endpoints. We also covered the incidence of these infections as well as the numerous
SC and
SC genotypes. The most important agents to treat cryptococcal infections are fluconazole (widely used), amphotericin B, and flucytosine. We provide data from the collaborative study that defined CLSI fluconazole ECVs for the most common cryptococcal species or genotypes and modes. EUCAST ECVs/ECOFFs are not yet available for fluconazole. We have summarized the incidence of cryptococccal infections (2000-2015) where fluconazole MICs were obtained by reference and commercial antifungal susceptibility tests. This occurrence is documented all over the world and those fluconazole MICs are mostly categorized by available CLSI ECVs/BPs as "resistant" instead of non-susceptible strains, including those by the commercial methods. As expected, the agreement between the CLSI and commercial methods is variable because SYO and Etest data could yield low/variable agreement (<90%) versus the CLSI method. Therefore, since BPs/ECVs are species and method dependent, why not gather sufficient MICs by commercial methods and define the required ECVs for these species?
Rice is a crucial crop to meet global food demands, therefore, it is necessary to develop rapid, efficient and inexpensive methods for screening mutants of important genes in molecular breeding. In ...this research, a simple microfluidic chip-integrated one-step polymerase chain reaction (PCR) and high-resolution melting (HRM) analysis were designed for the rapid screening of rice mutants. In order to complete the above processes, we also installed a self-developed functional instrument integrated with a temperature control system and a fluorescence detection department. This microfluidic platform was validated by analysing genes related to insect resistance, phenotype and yield of two important varieties of rice, Oryza sativa L. ssp. japonica. and Oryza sativa L. ssp. indica (93-11). The results showed that the platform could detect single-nucleotide mutations between the wild and the mutant rice plants in 1 h and the accuracy was consistent with gene sequencing regarded as the golden standard of the mutants screening. Compared to the traditional method (requiring approximately 3 h), this platform can distinguish mutants from wild types more rapidly. This system is a simple, integrated, disposable and low-cost tool for high-throughput screening of mutants in molecular breeding. More important, this portable instrument can be used as a point-of-care platform for nucleic acid diagnostics in many scientific fields.
New developments in HIV drug resistance CANE, Patricia A
Journal of antimicrobial chemotherapy,
09/2009, Volume:
64, Issue:
suppl-1
Journal Article, Conference Proceeding
Peer reviewed
Open access
Several new antiretroviral drugs have recently been licensed for use in HIV-1-infected patients. These include drugs in two new classes: an integrase inhibitor (raltegravir) and a CCR5 co-receptor ...antagonist (maraviroc). In addition, two new protease inhibitors, atazanavir and darunavir, which have activity against viruses resistant to other protease inhibitors, have come into clinical use. Finally etravirine, a novel non-nucleoside reverse transcriptase inhibitor (NNRTI) is being increasingly used in patients whose virus is resistant to the earlier NNRTIs. These clinical advances have required the development of novel assays and interpretation systems for detection of resistance to allow the laboratory monitoring of patients receiving these new therapies.
In a multicenter study a new, fully automated Roche Diagnostics Elecsys
®
HBsAg II screening assay with improved sensitivity to HBsAg mutant detection was compared to well-established HBsAg tests: ...AxSYM HBsAg V2 (Abbott), Architect HBsAg (Abbott), Advia Centaur HBsAg (Bayer) Enzygnost HBsAg 5.0 (Dade-Behring), and Vitros Eci HBsAg (Ortho). A total of 16 seroconversion panels, samples of 60 HBsAg native mutants, and 31 HBsAg recombinant mutants, dilution series of NIBSC and PEI standards, 156 HBV positive samples comprising genotypes A to G, 686 preselected HBsAg positive samples from different stages of infection, 3,593 samples from daily routine, and 6,360 unselected blood donations were tested to evaluate the analytical and clinical sensitivity, the detection of mutants, and the specificity of the new assay. Elecsys
®
HBsAg II showed a statistically significant better sensitivity in seroconversion panels to the compared tests. Fifty-seven out of 60 native mutants and all recombinant mutants were found positive. Among 156 HBV samples with different genotypes and 696 preselected HBsAg positive samples Elecsys
®
HBsAg II achieved a sensitivity of 100%. The lower detection limit for NIBSC standard was calculated to be 0.025 IU/ml and for the PEI standards ad and ay it was <0.001 and <0.005 U/ml, respectively. Within 2,724 daily routine specimens and 6.360 unselected blood donations Elecsys
®
HBsAg II showed a specificity of 99.97 and 99.88%, respectively. In conclusion the new Elecsys HBsAg II shows a high sensitivity for the detection of all stages of HBV infection and HBsAg mutants paired together with a high specificity in blood donors, daily routine samples, and potentially interfering sera.
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