Wild type and variants of SARS-COV-2 in Parisian sewage: presence in raw water and through processes in wastewater treatment plants.

The presence of SARS-CoV-2 RNA has been extensively reported at the influent of wastewater treatment plants (WWTPs) worldwide, and its monitoring has been proposed as a potential surveillance tool to early alert of epidemic outbreaks. However, the fate of the SARS-CoV-2 RNA in the treatment process of WWTP has not been widely studied yet; therefore, in this study, we aimed to evaluate the efficiency of treatment processes in reducing SARS-CoV-2 RNA levels in wastewater. The treatment process of three WWTPs of the Parisian area in France was monitored on six different weeks over a period of 2 months (from April 14 to June 9, 2021). SARS-CoV-2 RNA copies were detected using digital polymerase chain reaction (dPCR). Investigation on the presence of variants of concern (Del69-70, E484K, and L452R) was also performed. Additionally, SARS-CoV-2 RNA loads in the WWTPs influents were expressed as the viral concentration in per population equivalent (PE) and showed a good correlation with French public health indicators (incidence rate). SARS-CoV-2 RNA loads were notably reduced along the water treatment lines of the three WWTPs studied (2.5-3.4 log reduction). Finally, very low SARS-CoV-2 RNA loads were detected in effluents (non-detected in over half of the samples) which indicated that the potential risk of the release of wastewater effluents to the environment is probably insignificant, in the case of WWTPs enabling an efficient biological removal of nitrogen.

Efficient Genome Editing in Rice Protoplasts Using CRISPR/CAS9 Construct.

Genome editing technologies, mainly CRISPR/CAS9, are revolutionizing plant biology and breeding. Since the demonstration of its effectiveness in eukaryotic cells, a very large number of derived technologies has emerged. Demonstrating and comparing the effectiveness of all these new technologies in entire plants is a long, tedious, and labor-intensive process that generally involves the production of transgenic plants and their analysis. Protoplasts, plant cells free of their walls, offer a simple, high-throughput system to test the efficiency of these editing technologies in a few weeks' time span. We have developed a routine protocol using protoplasts to test editing technologies in rice. Our protocol allows to test more than 30 constructs in protoplasts prepared from leaf tissues of 100, 9-11-day-old seedlings. CRISPR/CAS9 construct effectiveness can be clearly established within less than a week. We provide here a full protocol, from designing sgRNA to mutation analysis.

A fast, efficient and high-throughput procedure involving laser microdissection and RT droplet digital PCR for tissue-specific expression profiling of rice roots.

BACKGROUND: In rice, the cortex and outer tissues play a key role in submergence tolerance. The cortex differentiates into aerenchyma, which are air-containing cavities that allow the flow of oxygen from shoots to roots, whereas exodermis suberification and sclerenchyma lignification limit oxygen loss from the mature parts of roots by forming a barrier to root oxygen loss (ROL). The genes and their networks involved in the cellular identity and differentiation of these tissues remain poorly understood. Identification and characterization of key regulators of aerenchyma and ROL barrier formation require determination of the specific expression profiles of these tissues. RESULTS: We optimized an approach combining laser microdissection (LM) and droplet digital RT-PCR (ddRT-PCR) for high-throughput identification of tissue-specific expression profiles. The developed protocol enables rapid (within 3 days) extraction of high-quality RNA from root tissues with a low contamination rate. We also demonstrated the possibility of extracting RNAs from paraffin blocks stored at 4 °C without any loss of quality. We included a detailed troubleshooting guide that should allow future users to adapt the proposed protocol to other tissues and/or species. We demonstrated that our protocol, which combines LM with ddRT-PCR, can be used as a complementary tool to in situ hybridization for tissue-specific characterization of gene expression even with a low RNA concentration input. We illustrated the efficiency of the proposed approach by validating three of four potential tissue-specific candidate genes detailed in the RiceXpro database. CONCLUSION: The detailed protocol and the critical steps required to optimize its use for other species will democratize tissue-specific transcriptome approaches combining LM with ddRT-PCR for analyses of plants.

Beyond Seek and Destroy: how to Generate Allelic Series Using Genome Editing Tools.

Genome editing tools have greatly facilitated the functional analysis of genes of interest by targeted mutagenesis. Many usable genome editing tools, including different site-specific nucleases and editor databases that allow single-nucleotide polymorphisms (SNPs) to be introduced at a given site, are now available. These tools can be used to generate high allelic diversity at a given locus to facilitate gene function studies, including examining the role of a specific protein domain or a single amino acid. We compared the effects, efficiencies and mutation types generated by our LbCPF1, SpCAS9 and base editor (BECAS9) constructs for the OsCAO1 gene. SpCAS9 and LbCPF1 have similar efficiencies in generating mutations but differ in the types of mutations induced, with the majority of changes being single-nucleotide insertions and short deletions for SpCAS9 and LbCPF1, respectively. The proportions of heterozygotes also differed, representing a majority in our LbCPF1, while with SpCAS9, we obtained a large number of biallelic mutants. Finally, we demonstrated that it is possible to specifically introduce stop codons using the BECAS9 with an acceptable efficiency of approximately 20%. Based on these results, a rational choice among these three alternatives may be made depending on the type of mutation that one wishes to introduce, the three systems being complementary. SpCAS9 remains the best choice to generate KO mutations in primary transformants, while if the desired gene mutation interferes with regeneration or viability, the use of our LbCPF1 construction will be preferred, because it produces mainly heterozygotes. LbCPF1 has been described in other studies as being as effective as SpCAS9 in generating homozygous and biallelic mutations. It will remain to be clarified in the future, whether the different LbCFP1 constructions have different efficiencies and determine the origin of these differences. Finally, if one wishes to specifically introduce stop codons, BECAS9 is a viable and efficient alternative, although it has a lower efficiency than SpCAS9 and LbCPF1 for creating KO mutations.