Role of phytohormones in regulating cold stress tolerance: Physiological and molecular approaches for developing cold-smart crop plants

Polymerase chain reaction (PCR)-based nucleic acid testing has played a critical role in disease diagnostics, pathogen surveillance, and many more. However, this method requires a long turnaround time, expensive equipment, and trained personnel, limiting its widespread availability and diagnostic capacity. On the other hand, the clustered regularly interspaced short palindromic repeats (CRISPR) technology has recently demonstrated capability for nucleic acid detection with high sensitivity and specificity. CRISPR-mediated biosensing holds great promise for revolutionizing nucleic acid testing procedures and developing point-of-care diagnostics. This review focuses on recent developments in both fundamental CRISPR biochemistry and CRISPR-based nucleic acid detection techniques. Four ongoing research hotspots in molecular diagnostics-target preamplification-free detection, microRNA (miRNA) detection, non-nucleic-acid detection, and SARS-CoV-2 detection-are also covered.

CRISPR-Cas Biochemistry and CRISPR-Based Molecular Diagnostics.

PAM-free loop-mediated isothermal amplification coupled with CRISPR/Cas12a cleavage (Cas-PfLAMP) for rapid detection of rice pathogens.

Abstract Anthocyanins are plant secondary metabolites that have a variety of biological functions, including pigmentation. The accumulation of anthocyanins is regulated by both transcriptional activators and repressors. Studies have shown that bZIP family members act primarily as positive regulators of anthocyanin biosynthesis, but there are few reports of negative regulation. Here, we report that a grapevine (Vitis vinifera) bZIP gene from group K, VvbZIP36, acts as a negative regulator of anthocyanin biosynthesis. Knocking out one allele of VvbZIP36 in grapevine with CRISPR/Cas9 promoted anthocyanin accumulation. Correlation analysis of transcriptome and metabolome data showed that a range of anthocyanin biosynthesis genes were activated in VvbZIP36 mutant plants relative to the wild type, resulting in the accumulation of related metabolites, including naringenin chalcone, naringenin, dihydroflavonols, and cyanidin-3-O-glucoside. The synthesis of stilbenes (α-viniferin), lignans, and some flavonols (including quercetin-3-O-rhamnoside, kaempferol-3-O-rhamnoside, and kaempferol-7-O-rhamnoside) was significantly inhibited, and several genes linked to their metabolism were downregulated in the mutant plants. In summary, our results demonstrate that VvbZIP36 is a negative regulator of anthocyanin biosynthesis that plays a role in balancing the synthesis of stilbenes (α-viniferin), lignans, flavonols, and anthocyanins.

/pdf/crispr-cas9-mediated-mutagenesis-of-vvbzip36-promotes-359cwktq.pdf

CRISPR/Cas9-mediated mutagenesis of VvbZIP36 promotes anthocyanin accumulation in grapevine (Vitis vinifera)

The global outbreak of the monkeypox virus (MPXV) highlights the need for rapid and cost‐effective MPXV detection tools to effectively monitor and control the monkeypox disease. Herein, we demonstrated a portable CRISPR–Cas‐based system for naked‐eye detection of MPXV. The system harnesses the high selectivity of CRISPR–Cas12 and the isothermal nucleic acid amplification potential of recombinase polymerase amplification. It can detect both the current circulating MPXV clade and the original clades. We reached a limit of detection (LoD) of 22.4 aM (13.5 copies/µl) using a microtiter plate reader, while the visual LoD of the system is 75 aM (45 copies/µl) in a two‐step assay, which is further reduced to 25 aM (15 copies/µl) in a one‐pot system. We compared our results with quantitative polymerase chain reaction and obtained satisfactory consistency. For clinical application, we demonstrated a sensitive and precise visual detection method with attomolar sensitivity and a sample‐to‐answer time of 35 min.

CRISPR–Cas12‐based field‐deployable system for rapid detection of synthetic DNA sequence of the monkeypox virus genome

https://doi.org/10.1016/j.bios.2022.113994

A one-pot CRISPR/Cas13a-based contamination-free biosensor for low-cost and rapid nucleic acid diagnostics

Cultivated grapevine (Vitis) is a highly valued horticultural crop, and cold stress affects its growth and productivity. Wild Amur grape (Vitis amurensis) PAT1 (Phytochrome A signal transduction 1, VaPAT1) is induced by low temperature, and ectopic expression of VaPAT1 enhances cold tolerance in Arabidopsis (Arabidopsis thaliana). However, little is known about the molecular mechanism of VaPAT1 during the cold stress response in grapevine. Here, we confirmed the overexpression of VaPAT1 in transformed grape calli enhanced cold tolerance. Yeast two-hybrid and bimolecular fluorescence complementation assays highlighted an interaction between VaPAT1 with INDETERMINATE-DOMAIN 3 (VaIDD3). A role of VaIDD3 in cold tolerance was also indicated. Transcriptome analysis revealed VaPAT1 and VaIDD3 overexpression and cold treatment coordinately modulate the expression of stress-related genes including lipoxygenase 3 (LOX3), a gene encoding a key jasmonate biosynthesis enzyme. Co-expression network analysis indicated LOX3 might be a downstream target of VaPAT1. Both electrophoretic mobility shift and dual luciferase reporter assays showed the VaPAT1-IDD3 complex binds to the IDD-box (AGACAAA) in the VaLOX3 promoter to activate its expression. Overexpression of both VaPAT1 and VaIDD3 increased the transcription of VaLOX3 and JA levels in transgenic grape calli. Conversely, VaPAT1-SRDX (dominant repression) and CRISPR/Cas9-mediated mutagenesis of PAT1-ED causing the loss of the C-terminus in grape calli dramatically prohibited the accumulation of VaLOX3 and JA levels during cold treatment. Together, these findings point to a pivotal role of VaPAT1 in the cold stress response in grape by regulating JA biosynthesis.

GRAS-domain transcription factor PAT1 regulates jasmonic acid biosynthesis in grape cold stress response.

The efficacy of the CRISPR/Cas9 system in grapevine (Vitis vinifera L.) has been documented, but the optimization of this system, as well as CRISPR/Cas9-mediated multiplex genome editing, has not been explored in this species. Herein, we identified four VvU3 and VvU6 promoters and two ubiquitin (UBQ) promoters in grapevine and demonstrated that the use of the identified VvU3/U6 and UBQ2 promoters could significantly increase the editing efficiency in grape by improving the expression of sgRNA and Cas9, respectively. Furthermore, we conducted multiplex genome editing using the optimized CRISPR/Cas9 vector that contained the conventional multiple sgRNA expression cassettes or the polycistronic tRNA-sgRNA cassette (PTG) by targeting the sugar-related tonoplastic monosaccharide transporter (TMT) family members TMT1 and TMT2, and the overall editing efficiencies were higher than 10%. The simultaneous editing of TMT1 and TMT2 resulted in reduced sugar levels, which indicated the role of these two genes in sugar accumulation in grapes. Moreover, the activities of the VvU3, VvU6, and UBQ2 promoters in tobacco genome editing were demonstrated by editing the phytoene desaturase (PDS) gene in Nicotiana benthamiana leaves. Our study provides materials for the optimization of the CRISPR/Cas9 system. To our knowledge, our simultaneous editing of the grape TMT family genes TMT1 and TMT2 constitutes the first example of multiplex genome editing in grape. The multiplex editing systems described in this manuscript expand the toolbox of grape genome editing, which would facilitate basic research and molecular breeding in grapevine.

/pdf/optimizing-the-crispr-cas9-system-for-genome-editing-in-17n1va1ab0.pdf

Optimizing the CRISPR/Cas9 system for genome editing in grape by using grape promoters.

Abstract Overexpression and knockout (or knockdown) of gene of interest are two commonly used strategies for gene functional study. Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system-mediated gene knockout has been applied in most plant species, including grapevine. However, CRISPR/dCas9 (deactivated Cas9)-based transcriptional activation is still unreported in fruit crops, although a few studies have been documented in Arabidopsis and rice. Here, we tested two transcriptional activators VP64 and TV for transcriptional activation of endogenous genes in grape. Both the dCas9-VP64 and dCas9-TV systems are efficient enough for transcriptional activation of the UDP-glucose flavonoid glycosyltransferases (UFGT) gene in grape cells. The effectiveness of the dCas9-VP64 system in UFGT activation was about 1.6- to 5.6-fold, while the efficiency of the dCas9-TV system was around 5.7- to 7.2-fold. Moreover, in grapevine plants, highly efficient activation of the cold-responsive transcription factor gene CBF4 was achieved by using the dCas9-TV system. The expression of CBF4 was increased 3.7- to 42.3-fold in transgenic plants. Compared with the wild-type plants, the CBF4-activated plants exhibited lower electrolyte leakage after cold treatment. Our results demonstrate the effectiveness of the dCas9-VP64 and dCas9-TV systems in gene activation in grape, which will facilitate application of transcriptional activation in this economically important species.

https://academic.oup.com/hr/article-pdf/doi/10.1093/hr/uhab037/43336531/uhab037.pdf

Highly efficient activation of endogenous gene in grape using CRISPR/dCas9-based transcriptional activators

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A one-pot CRISPR/Cas13a-based contamination-free biosensor for low-cost and rapid nucleic acid diagnostics

GRAS-domain transcription factor PAT1 regulates jasmonic acid biosynthesis in grape cold stress response.

Optimizing the CRISPR/Cas9 system for genome editing in grape by using grape promoters.

Highly efficient activation of endogenous gene in grape using CRISPR/dCas9-based transcriptional activators