Cas-OPRAD: a one-pot RPA/PCR CRISPR/Cas12 assay for on-site Phytophthora root rot detection.

Phytophthora sojae is a devastating plant pathogen that causes soybean Phytophthora root rot worldwide. Early on-site and accurate detection of the causal pathogen is critical for successful management. In this study, we have developed a novel and specific one-pot RPA/PCR-CRISPR/Cas12 assay for on-site detection (Cas-OPRAD) of Phytophthora root rot (P. sojae). Compared to the traditional RPA/PCR detection methods, the Cas-OPRAD assay has significant detection performance. The Cas-OPRAD platform has excellent specificity to distinguish 33 P. sojae from closely related oomycetes or fungal species. The PCR-Cas12a assay had a consistent detection limit of 100 pg. µL-1, while the RPA-Cas12a assay achieved a detection limit of 10 pg. µL-1. Furthermore, the Cas-OPRAD assay was equipped with a lateral flow assay for on-site diagnosis and enabled the visual detection of P. sojae on the infected field soybean samples. This assay provides a simple, efficient, rapid (<1 h), and visual detection platform for diagnosing Phytophthora root rot based on the one-pot CRISPR/Cas12a assay. Our work provides important methods for early and accurate on-site detection of Phytophthora root rot in the field or customs fields.

An ultra-sensitive one-pot RNA-templated DNA ligation rolling circle amplification-assisted CRISPR/Cas12a detector assay for rapid detection of SARS-CoV-2.

Rapid, sensitive, and one-pot diagnosis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) plays an extremely important role in point-of-care testing (POCT). Herein, we report an ultra-sensitive and rapid one-pot enzyme-catalyzed rolling circle amplification-assisted CRISPR/FnCas12a assay, termed OPERATOR. OPERATOR employs a single well-designed single-strand padlock DNA, containing a protospacer adjacent motif (PAM) site and a sequence complementary to the target RNA which procedure converts and amplifies genomic RNA to DNA by RNA-templated DNA ligation and multiply-primed rolling circle amplification (MRCA). The MRCA amplicon of single-stranded DNA is cleaved by the FnCas12a/crRNA complex and detected via a fluorescence reader or lateral flow strip. OPERATOR presents outstanding advantages including ultra-sensitivity (1.625 copies per reaction), high specificity (100%), rapid reaction speed (∼30 min), easy operation, low cost, and on-spot visualization. Furthermore, we established a POCT platform by combining OPERATOR with rapid RNA release and a lateral flow strip without professional equipment. The high performance of OPERATOR in SARS-CoV-2 tests was confirmed using both reference materials and clinical samples, and the results suggest that is readily adaptable for point-of-care testing of other RNA viruses.

One Versatile Cas9-Integrated Single-Tube Duplex Quantitative Real-Time PCR System for Rapid Analysis of CRISPR/Cas-Induced Mutants.

Clustered regularly interspersed short palindromic repeat (CRISPR)/Cas9 gene editing has become a common tool for rapid crop and animal breeding, but efficiently screening out and genotyping for the CRISPR/Cas9-induced mutant lines at a low cost remains challenging. Using rice (Oryza sativa L.) samples genetically edited at the Waxy locus as an example, we developed a single-tube duplex quantitative real-time PCR assisted by an in vitro CRISPR/Cas9 cleavage (Cc-qPCR) method to screen for expected genetically edited lines, identify genotypes, and evaluate gene-editing frequency. In Cc-qPCR, genomic DNA is first cleaved at the target site by the single-guide RNA (sgRNA)/Cas9 complex and then quantified with qPCR to assess for the presence of a mutant and identify sample genotypes. Our findings suggest that Cc-qPCR can successfully identify mutants with small insertions or deletions (indels), even in mutant lines with single-base indels or substitutions. Cc-qPCR was also able to successfully identify heterozygous and homozygous mutants. The sensitivity of Cc-qPCR was determined to be as low as 0.5%, indicating that the method could be used to evaluate the editing efficiency of gene-editing systems. After testing our novel method on Waxy locus-edited rice offspring, our results show that Cc-qPCR is an accurate and effective approach to rapidly identify expected mutants and their genotypes and to evaluate editing efficiency. This method will prove useful for increasing the efficiency and range of molecular breeding techniques.

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

Pathogenic disease is an important factor affecting rice growth, yield and quality, and the development and application of rapid diagnostic methods will contribute to the prevention and control of rice disease. Herein, we developed a novel protospacer adjacent motif (PAM)-free loop-mediated isothermal amplification (LAMP) assisted CRISPR/Cas12a cleavage (Cas-PfLAMP) assay for detection of three rice pathogens; Xanthomonas oryzae pv. Oryzae (XOO), rice stripe virus (RSV), and rice black-streaked dwarf virus (RBSDV). The Cas-PfLAMP assay showed high specificity due to doubly specific recognition of LAMP primer sets and FnCas12a/sgRNA, and high sensitivity down to 9 or 3 copies due to LAMP amplification and CRISPR/Cas12a trans cleavage activity. Furthermore, a visual on-spot Cas-PfLAMP platform was established for detection of rice pathogens by combining solid-phase nucleic acid extraction and a lateral flow strip (LFS) test. Analysis of rice leaf field samples confirmed the impressive performance of the Cas-PfLAMP platform, demonstrating its suitability for rapid (∼50 min) on-spot detection of rice diseases. The assay could also be extended to detection of other plant diseases, and other nucleic acid field tests.

Whole-Genome Sequence of Synthesized Allopolyploids in Cucumis Reveals Insights into the Genome Evolution of Allopolyploidization.

The importance of allopolyploidy in plant evolution has been widely recognized. The genetic changes triggered by allopolyploidy, however, are not yet fully understood due to inconsistent phenomena reported across diverse species. The construction of synthetic polyploids offers a controlled approach to systematically reveal genomic changes that occur during the process of polyploidy. This study reports the first fully sequenced synthetic allopolyploid constructed from a cross between Cucumis sativus and C. hystrix, with high-quality assembly. The two subgenomes are confidently partitioned and the C. sativus-originated subgenome predominates over the C. hystrix-originated subgenome, retaining more sequences and showing higher homeologous gene expression. Most of the genomic changes emerge immediately after interspecific hybridization. Analysis of a series of genome sequences from several generations (S0, S4-S13) of C. ×hytivus confirms that genomic changes occurred in the very first generations, subsequently slowing down as the process of diploidization is initiated. The duplicated genome of the allopolyploid with double genes from both parents broadens the genetic base of C. ×hytivus, resulting in enhanced phenotypic plasticity. This study provides novel insights into plant polyploid genome evolution and demonstrates a promising strategy for the development of a wide array of novel plant species and varieties through artificial polyploidization.

Global Profiling of lncRNAs Expression Responsive to Allopolyploidization in Cucumis.

Long non-coding RNAs (lncRNAs) play critical regulatory roles in various biological processes. However, the presence of lncRNAs and how they function in plant polyploidy are still largely unknown. Hence, we examined the profile of lncRNAs in a nascent allotetraploid Cucumis hytivus (S14), its diploid parents, and the F1 hybrid, to reveal the function of lncRNAs in plant-interspecific hybridization and whole genome duplication. Results showed that 2206 lncRNAs evenly transcribed from all 19 chromosomes were identified in C. hytivus, 44.6% of which were from intergenic regions. Based on the expression trend in allopolyploidization, we found that a high proportion of lncRNAs (94.6%) showed up-regulated expression to varying degrees following hybridization. However, few lncRNAs (33, 2.1%) were non-additively expressed after genome duplication, suggesting the significant effect of hybridization on lncRNAs, rather than genome duplication. Furthermore, 253 cis-regulated target genes were predicted for these differentially expressed lncRNAs in S14, which mainly participated in chloroplast biological regulation (e.g., chlorophyll synthesis and light harvesting system). Overall, this study provides new insight into the function of lncRNAs during the processes of hybridization and polyploidization in plant evolution.

Nuclear-Cytoplasmic Coevolution Analysis of RuBisCO in Synthesized Cucumis Allopolyploid.

Allopolyploids are often faced with the challenge of maintaining well-coordination between nuclear and cytoplasmic genes inherited from different species. The synthetic allotetraploid Cucumis × hytivus is a useful model to explore cytonuclear coevolution. In this study, the sequences and expression of cytonuclear enzyme complex RuBisCO as well as its content and activity in C. × hytivus were compared to its parents to explore plastid-nuclear coevolution. The plastome-coded rbcL gene sequence was confirmed to be stable maternal inheritance, and parental copy of nuclear rbcS genes were both preserved in C. × hytivus. Thus, the maternal plastid may interact with the biparentally inherited rbcS alleles. The expression of the rbcS gene of C-homoeologs (paternal) was significantly higher than that of H-homoeologs (maternal) in C. × hytivus (HHCC). Protein interaction prediction analysis showed that the rbcL protein has stronger binding affinity to the paternal copy of rbcS protein than that of maternal copy in C. × hytivus, which might explain the transcriptional bias of the rbcS homoeologs. Moreover, both the activity and content of RuBisCO in C. × hytivus showed mid-parent heterosis. In summary, our results indicate a paternal transcriptional bias of the rbcS genes in C. × hytivus, and we found new nuclear-cytoplasmic combination may be one of the reasons for allopolyploids heterosis.

Allopolyploidization in Cucumis contributes to delayed leaf maturation with repression of redundant homoeologous genes.

The important role of polyploidy in plant evolution is widely recognized. However, many questions remain to be explored to address how polyploidy affects the phenotype of the plant. To shed light on the phenotypic and molecular impacts of allopolyploidy, we investigated the leaf development of a synthesized allotetraploid (Cucumis × hytivus), with an emphasis on chlorophyll development. Delayed leaf maturation was identified in C. × hytivus, based on delayed leaf expansion, initial chlorophyll deficiency in the leaves and disordered sink-source transition. Anatomical observations also revealed disturbed chloroplast development in C. ×hytivus. The determination of chlorophyll biosynthesis intermediates suggested that the chlorophyll biosynthesis pathway of C. × hytivus is blocked at the site at which uroporphyrinogen III is catalysed to coproporphyrinogen III. Three chlorophyll biosynthesis-related genes, HEMA1, HEME2 and POR, were significantly repressed in C. × hytivus. Sequence alignment showed both synonymous and non-synonymous substitutions in the HEMA1, HEME2 and POR genes of the parents. Cloning of the chlorophyll biosynthetic genes suggested the retention of homoeologs. In addition, a chimeric clone of the HEMA1 gene that consisted of homologous genes from the parents was identified in C. × hytivus. Overall, our results showed that allopolyploidization in Cucumis has resulted in disturbed chloroplast development and reduced chlorophyll biosynthesis caused by the repressed expression of duplicated homologous genes, which further led to delayed leaf maturation in the allotetraploid, C. × hytivus. The preferential retention/loss of certain types of genes and non-reciprocal homoeologous recombination were also supported in the present study, which provides new insights into the impact of allopolyploidy.