Implementation of the CRISPR-Cas13a system in fission yeast and its repurposing for precise RNA editing.

In contrast to genome editing, which introduces genetic changes at the DNA level, disrupting or editing gene transcripts provides a distinct approach to perturbing a genetic system, offering benefits complementary to classic genetic approaches. To develop a new toolset for manipulating RNA, we first implemented a member of the type VI CRISPR systems, Cas13a from Leptotrichia shahii (LshCas13a), in Schizosaccharomyces pombe, an important model organism employed by biologists to study key cellular mechanisms conserved from yeast to humans. This approach was shown to knock down targeted endogenous gene transcripts with different efficiencies. Second, we engineered an RNA editing system by tethering an inactive form of LshCas13a (dCas13) to the catalytic domain of human adenosine deaminase acting on RNA type 2 (hADAR2d), which was shown to be programmable with crRNA to target messenger RNAs and precisely edit specific nucleotide residues. We optimized system parameters using a dual-fluorescence reporter and demonstrated the utility of the system in editing randomly selected endogenous gene transcripts. We further used it to restore the transposition of retrotransposon Tf1 mutants in fission yeast, providing a potential novel toolset for retrovirus manipulation and interference.

Revealing Differential RNA Editing Specificity of Human ADAR1 and ADAR2 in Schizosaccharomyces pombe.

Adenosine-to-inosine (A-to-I) RNA editing is an important post-transcriptional modification mediated by the adenosine deaminases acting on RNA (ADAR) family of enzymes, expanding the transcriptome by altering selected nucleotides A to I in RNA molecules. Recently, A-to-I editing has been explored for correcting disease-causing mutations in RNA using therapeutic guide oligonucleotides to direct ADAR editing at specific sites. Humans have two active ADARs whose preferences and specificities are not well understood. To investigate their substrate specificity, we introduced hADAR1 and hADAR2, respectively, into Schizosaccharomyces pombe (S. pombe), which lacks endogenous ADARs, and evaluated their editing activities in vivo. Using transcriptome sequencing of S. pombe cultured at optimal growth temperature (30 °C), we identified 483 A-to-I high-confident editing sites for hADAR1 and 404 for hADAR2, compared with the non-editing wild-type control strain. However, these sites were mostly divergent between hADAR1 and hADAR2-expressing strains, sharing 33 common sites that are less than 9% for each strain. Their differential specificity for substrates was attributed to their differential preference for neighboring sequences of editing sites. We found that at the -3-position relative to the editing site, hADAR1 exhibits a tendency toward T, whereas hADAR2 leans toward A. Additionally, when varying the growth temperature for hADAR1- and hADAR2-expressing strains, we observed increased editing sites for them at both 20 and 35 °C, compared with them growing at 30 °C. However, we did not observe a significant shift in hADAR1 and hADAR2's preference for neighboring sequences across three temperatures. The vast changes in RNA editing sites at lower and higher temperatures were also observed for hADAR2 previously in budding yeast, which was likely due to the influence of RNA folding at these different temperatures, among many other factors. We noticed examples of longer lengths of dsRNA around the editing sites that induced editing at 20 or 35 °C but were absent at the other two temperature conditions. We found genes' functions can be greatly affected by editing of their transcripts, for which over 50% of RNA editing sites for both hADAR1 and hADAR2 in S. pombe were in coding sequences (CDS), with more than 60% of them resulting in amino acid changes in protein products. This study revealed the extensive differences in substrate selectivity between the two active human ADARS, i.e., ADAR1 and ADAR2, and provided novel insight when utilizing the two different enzymes for in vivo treatment of human genetic diseases using the RNA editing approach.

DeepEdit: single-molecule detection and phasing of A-to-I RNA editing events using nanopore direct RNA sequencing.

Single-molecule detection and phasing of A-to-I RNA editing events remain an unresolved problem. Long-read and PCR-free nanopore native RNA sequencing offers a great opportunity for direct RNA editing detection. Here, we develop a neural network model, DeepEdit, that not only recognizes A-to-I editing events in single reads of Oxford Nanopore direct RNA sequencing, but also resolves the phasing of RNA editing events on transcripts. We illustrate the robustness of DeepEdit by applying it to Schizosaccharomyces pombe and Homo sapiens transcriptome data. We anticipate DeepEdit to be a powerful tool for the study of RNA editing from a new perspective.

Multi-valent mRNA vaccines against monkeypox enveloped or mature viron surface antigens demonstrate robust immune response and neutralizing activity.

Monkeypox was declared a global health emergency by the World Health Organization, and as of March 2023, 86,000 confirmed cases and 111 deaths across 110 countries have been reported. Its causal agent, monkeypox virus (MPV) belongs to a large family of double-stranded DNA viruses, Orthopoxviridae, that also includes vaccinia virus (VACV) and others. MPV produces two distinct forms of viral particles during its replication cycles: the enveloped viron (EV) that is released via exocytosis, and the mature viron (MV) that is discharged through lysis of host cells. This study was designed to develop multi-valent mRNA vaccines against monkeypox EV and MV surface proteins, and examine their efficacy and mechanism of action. Four mRNA vaccines were produced with different combinations of surface proteins from EV (A35R and B6R), MV (A29L, E8L, H3L and M1R), or EV and MV, and were administered in Balb/c mice to assess their immunogenicity potentials. A dynamic immune response was observed as soon as seven days after initial immunization, while a strong IgG response to all immunogens was detected with ELISA after two vaccinations. The higher number of immunogens contributed to a more robust total IgG response and correlating neutralizing activity against VACV, indicating the additive potential of each immunogen in generating immune response and nullifying VACV infection. Further, the mRNA vaccines elicited an antigen-specific CD4+ T cell response that is biased towards Th1. The mRNA vaccines with different combinations of EV and MV surface antigens protected a mouse model from a lethal dose VACV challenge, with the EV and MV antigens-combined vaccine offering the strongest protection. These findings provide insight into the protective mechanism of multi-valent mRNA vaccines against MPV, and also the foundation for further development of effective and safe mRNA vaccines for enhanced protection against monkeypox virus outbreak.

Targeted inhibition of Zika virus infection in human cells by CRISPR-Cas13b.

Zika virus (ZIKV) outbreaks occurred in recent years on an unprecedented scale, which caused fever and severe complications like Guillain-Barré syndrome in adults and fetal abnormalities. No vaccines or other effective treatments against ZIKV are available to date. The CRISPR-Cas13 family has the unique ability to target single-strand RNA molecules and mediate RNA cleavage. In the present study, we sought to exploit CRISPR-Cas13b for developing an anti-ZIKV system in mammalian cells. We first generated a ZIKV infection and reporting system by: (1) fusing mCherry to the ZIKV capsid protein for reporting infection by fluorescence; and (2) deriving a 293T cell line (293T-DC-SIGN) stably expressing DC-SIGN (Dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin) that became highly susceptible to ZIKV infection. The CRISPR Cas13b expression was reported to be in the cytoplasm of 293T-DC-SIGN cells using a Cas13b-GFP fusion expression vector. Fourteen CRISPR RNAs (crRNAs) were designed to target the most conserved regions of the ZIKV genome through bioinformatics analysis of 1138 ZIKV genome sequences. Five crRNAs were found to have significant effects (p < 0.001; two-sided t test) for Cas13b-targeted inhibition on ZIKV infection in 293T-DC-SIGN cells. Our study demonstrated an exciting example of using the CRISPR-Cas13b system for the treatment and prevention of ZIKV infection, highlighting CRISPR-Cas13 as a promising therapeutic anti-RNA virus strategy.

Interfering with retrotransposition by two types of CRISPR effectors: Cas12a and Cas13a.

CRISPRs are a promising tool being explored in combating exogenous retroviral pathogens and in disabling endogenous retroviruses for organ transplantation. The Cas12a and Cas13a systems offer novel mechanisms of CRISPR actions that have not been evaluated for retrovirus interference. Particularly, a latest study revealed that the activated Cas13a provided bacterial hosts with a "passive protection" mechanism to defend against DNA phage infection by inducing cell growth arrest in infected cells, which is especially significant as it endows Cas13a, a RNA-targeting CRISPR effector, with mount defense against both RNA and DNA invaders. Here, by refitting long terminal repeat retrotransposon Tf1 as a model system, which shares common features with retrovirus regarding their replication mechanism and life cycle, we repurposed CRISPR-Cas12a and -Cas13a to interfere with Tf1 retrotransposition, and evaluated their different mechanisms of action. Cas12a exhibited strong inhibition on retrotransposition, allowing marginal Tf1 transposition that was likely the result of a lasting pool of Tf1 RNA/cDNA intermediates protected within virus-like particles. The residual activities, however, were completely eliminated with new constructs for persistent crRNA targeting. On the other hand, targeting Cas13a to Tf1 RNA intermediates significantly inhibited Tf1 retrotransposition. However, unlike in bacterial hosts, the sustained activation of Cas13a by Tf1 transcripts did not cause cell growth arrest in S. pombe, indicating that virus-activated Cas13a likely acted differently in eukaryotic cells. The study gained insight into the actions of novel CRISPR mechanisms in combating retroviral pathogens, and established system parameters for developing new strategies in treatment of retrovirus-related diseases.