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Tools & Techniques: microRNAs for Biosafety; Genome-wide RNAi Mutant Library in Rice; and More

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Looking for a way to reduce the risk of human infection in influenza gain-of-function studies, Mount Sinai researchers have developed a microRNA-based approach to boost biosafety that they say can be applied to a number of other pathogenic viruses.

Although the H5N1 flu strain is primarily limited to migratory birds, the virus has killed several hundred humans over the past 15 years, making continued research a necessity.

However, while infected humans have heretofore acquired the virus from direct contact with birds, recent gain-of-function experiments indicate that as few as three amino acid substitutions are sufficient to enable the airborne transmission of certain H5N1 strains between ferrets — a surrogate indicator of transmission between humans, the researchers wrote in Nature Biotechnology.

“These findings raised serious public concern about whether the scientific community should be generating mammalian transmissible influenza A viruses, even within the controlled setting of enhanced biosafety level 3 containment,” they noted in their paper.

To address this issue, the scientists proposed a microRNA-based strategy to mitigate the risk of such gain-of-function flu studies.

Having previously demonstrated that miRNA-binding sites engineered into the influenza A virus genome could suppress viral gene expression and infection, they used endogenous miRNAs to limit viral tropism.

The team found that miR-192 is expressed in primary human respiratory tract epithelial cells, as well as in mouse lungs, but absent from the ferret respiratory tract. Incorporating miR-192 target sites into the influenza A virus did not prevent flu replication and transmissibility in ferrets, they found, but “did attenuate influenza pathogenicity in mice,” according to the Nature Biotechnology paper.

Importantly, this strategy does not appear limited to influenza A, they concluded, but could be used to minimize the risks associated with gain-of-function experiments of other highly pathogenic viruses such as Ebola.

“The only requirements for this approach are miRNAs that are absent in cells of the model system where replication is meant to occur but present in human cells, and a genetic system to permit the insertion of miRNA target sites into the genome,” they noted.


Although long hairpin RNA transgenes are effective RNAi tools for studying gene function in plants, a genome-wide RNAi mutant collection using hpRNA transgenes has not been reported for plants.

To fill this gap, scientists from the Chinese Academy of Agricultural Sciences and from Australia’s Commonwealth Scientific and Industrial Research Organization have developed an hpRNA library for the genome-wide identification of gene function in rice using a novel rolling circle amplification-mediated hpRNA method.

According to a report in the Plant Biotechnology Journal, “transformation of rice with the library resulted in thousands of transgenic lines containing hpRNAs targeting genes of various function.”

Target mRNA was downregulated in the hpRNA lines, which correlated with the accumulation of siRNAs corresponding to the double-stranded arms of the hpRNA. Multiple members of a gene family were simultaneously silenced by hpRNAs derived from a single member, but the degree of cross-silencing depended on the level of sequence homology between members and the abundance of matching siRNAs.

“The silencing of key genes tended to cause a severe phenotype, but these transgenic lines usually survived in the field long enough for phenotypic and molecular analyses to be conducted,” they added. “Deep sequencing analysis of small RNAs showed that the hpRNA-derived siRNAs were characteristic of Argonaute-binding small RNAs.”


As research into miRNAs continues, recent reports have demonstrated the existence of miRNA isoforms — miRNAs with sequences that vary from reference miRNA sequences.

In order to improve researchers’ ability to identify these so-called isomiRs, a team from the Federal University of Rio Grande do Sul has created a tool called isomiRID that standardizes and automates the search for isoforms in high-throughput small RNA sequencing libraries.

The classification of isomiRs centers around three major categories: 5’, 3’, and polymorphic isomiRs, with the subclassification of 5’ and 3’ isomiRs into templated or nontemplated modifications according to the miRNA precursor sequence, the scientists wrote in Bioinformatics.

“Nucleotide additions at the 5’ end have also been reported altering the miRNA seed region and consequently its functionality,” they wrote. Additionally, “several isomiR variants have been discovered with deep sequencing technologies.”

According to the paper, isomiRID allows for the identification of 5’, 3’, and polymorphic isomiRs based on the canonical miRNA known sequence, as well as from other regions on the same miRNA precursor. “The program can also identify nontemplated 5’ or 3’ end variations by mapping the sRNAs in the known pre-miRNAs.”

The tool can be accessed here.


Continuing their work on single-stranded siRNAs, researchers from Isis Pharmaceuticals and the University of Texas Southwestern Medical Center have reported on the development of such RNAi molecules capable of inhibiting ataxin-3 expression in vitro.

Previously, the scientists had published data showing that ss-siRNAs could be used to silence expression of the huntingtin protein, a target in treating Huntington’s disease. Building off that work, they tested the ss-siRNAs against the mutant form of ataxin-3 — the genetic cause of Machado-Joseph disease, which, like Huntington’s disease, is caused by expansion of a CAG repeat.

“One goal for these studies was to examine the hypothesis that a single anti-CAG ss-siRNA can be a lead compound for developing therapies for multiple trinucleotide expansion diseases by expanding inhibition” to ataxin-3, they wrote in Nucleic Acids Research. “A second goal was to understand similarities and differences between duplex RNAs and ss-siRNAs during allele-selective inhibition of gene expression.”

They found that their RNAi molecules were capable of selectively blocking expression of mutant ataxin-3 but, importantly, through an RNAi mechanism, as well as RNAi-independent manner that alters splicing of the protein.

“Our data suggest that the RNAi-independent steric-blocking mechanism can prevail for some ss-siRNAs for inhibiting [ataxion-3] expression,” they wrote. “This mechanism reduces allele selectivity and leads to formation of a higher mobility product.”

The researchers note that their work with ss-siRNAs is at an “early stage,” but state that their results to date suggest that the molecules can be tailored to silence gene expression in different ways, improving their potential for drug discovery.

The Scan

Push Toward Approval

The Wall Street Journal reports the US Food and Drug Administration is under pressure to grant full approval to SARS-CoV-2 vaccines.

Deer Exposure

About 40 percent of deer in a handful of US states carry antibodies to SARS-CoV-2, according to Nature News.

Millions But Not Enough

NPR reports the US is set to send 110 million SARS-CoV-2 vaccine doses abroad, but that billions are needed.

PNAS Papers on CRISPR-Edited Cancer Models, Multiple Sclerosis Neuroinflammation, Parasitic Wasps

In PNAS this week: gene-editing approach for developing cancer models, role of extracellular proteins in multiple sclerosis, and more.