CRISPR/Cas13 effectors have differing extents of off-target effects that limit their utility in eukaryotic cells

CRISPR/Cas13 effectors have garnered increasing attention as easily customizable tools for detecting and depleting RNAs of interest. Near perfect complementarity between a target RNA and the Cas13-associated guide RNA is required for activation of Cas13 ribonuclease activity. Nonetheless, the specificity of Cas13 effectors in eukaryotic cells has been debated as the Cas13 nuclease domains can be exposed on the enzyme surface, providing the potential for promiscuous cleavage of nearby RNAs (so-called collateral damage). Here, using co-transfection assays in Drosophila and human cells, we found that the off-target effects of RxCas13d, a commonly used Cas13 effector, can be as strong as the level of on-target RNA knockdown. The extent of off-target effects is positively correlated with target RNA expression levels, and collateral damage can be observed even after reducing RxCas13d/guide RNA levels. The PspCas13b effector showed improved specificity and, unlike RxCas13d, can be used to deplete a Drosophila circular RNA without affecting the expression of the associated linear RNA. PspCas13b nonetheless still can have off-target effects and we notably found that the extent of off-target effects for Cas13 effectors differs depending on the cell type and target RNA examined. In total, these results highlight the need for caution when designing and interpreting Cas13-based knockdown experiments.

Mirtron-mediated RNA knockdown/replacement therapy for the treatment of dominant retinitis pigmentosa

Rhodopsin (RHO) gene mutations are a common cause of autosomal dominant retinitis pigmentosa (ADRP). The need to suppress toxic protein expression together with mutational heterogeneity pose challenges for treatment development. Mirtrons are atypical RNA interference effectors that are spliced from transcripts as short introns. Here, we develop a novel mirtron-based knockdown/replacement gene therapy for the mutation-independent treatment of RHO-related ADRP, and demonstrate efficacy in a relevant mammalian model. Splicing and potency of rhodopsin-targeting candidate mirtrons are initially determined, and a mirtron-resistant codon-modified version of the rhodopsin coding sequence is validated in vitro. These elements are then combined within a single adeno-associated virus (AAV) and delivered subretinally in a RhoP23H knock-in mouse model of ADRP. This results in significant mouse-to-human rhodopsin RNA replacement and is associated with a slowing of retinal degeneration. This provides proof of principle that synthetic mirtrons delivered by AAV are capable of reducing disease severity in vivo.

A programmable probiotic encapsulation system enhances therapeutic delivery in vivo

Recent advances in therapeutic modulation of human microbiota have driven new efforts to engineer living microbial medicines using synthetic biology. However, a long-standing challenge for live bacterial therapies is balancing the high dose required to achieve robust efficacy with the potential for sepsis. Here, we developed a genetically encoded microbial encapsulation system with tunable and dynamic expression of surface capsular polysaccharides to enhance therapeutic delivery. Following a synthetic small RNA knockdown screen of the capsular biosynthesis pathway, we constructed synthetic gene circuits that regulate bacterial encapsulation based on sensing the levels of environmental inducer, bacterial density, and blood pH. The induced encapsulation system enabled tunable immunogenicity and survivability of the probiotic Escherichia coli, resulting in increased maximum tolerated dose and enhanced efficacy in murine cancer models. Furthermore, triggering in situ encapsulation was found to increase microbial translocation between mouse tumors, leading to efficacy in distal tumors. The programmable encapsulation system demonstrates a new approach to control microbial therapeutic profiles in vivo using synthetic biology.

Efficient RNA virus targeting via CRISPR-CasRx in fish

The emergence of the CRISPR-Cas system as a technology has transformed our ability to modify nucleic acids, and the CRISPR-Cas13 system has been used to target RNA. CasRx is a small sized type VI-D effector (Cas13d) with RNA knockdown efficiency that may have an interference effect on RNA viruses. However, the RNA virus-targeting activity of CasRx still needs to be verified in vivo in vertebrates. In this study, we successfully engineered a highly effective CasRx system for fish virus interference. We designed synthetic mRNA coding for CasRx and used CRISPR RNAs to guide it to target the grouper nervous necrosis virus (RGNNV). This technique resulted in significant interference with virus infections both in vitro and in vivo . These results indicate that CRISPR/CasRx can be used to engineer interference against RNA viruses in fish, which provides a potential novel mechanism for RNA-guided immunity against other RNA viruses in vertebrates. Importance RNA viruses are most important viral pathogens infecting vertebrates and mammals. RNA virus populations are highly dynamic due to short generation times, large population sizes, and high mutation frequencies. Therefore, it is difficult to find a widely effective ways to inhibit RNA viruses. Therefore, we urgently need to develop effective antiviral methods. CasRx is a small sized type VI-D effector (Cas13d) with RNA knockdown efficiency that can have an interference effect on RNA viruses. Nervous necrosis virus (NNV), a non-enveloped positive-strand RNA virus, is one of the most serious viral pathogens infecting more than 40 cultured fish species resulting in huge economic losses worldwide. Here, we establish a novel efective CasRx system for RNA virus interference using NNV and grouper (Epinephelus coioices) as model. Our data show that CasRx have the most robust for RNA virus interference applications in fish and demonstrate its suitability for studying key questions relating to virus biology.

CRISPR/Cas13d mediated efficient KDM5B mRNA knockdown in porcine cells and parthenogenetic embryos

An efficient mRNA knockdown strategy is needed to explore gene function in cells and embryos, especially to understand the process of maternal mRNA decay during early embryo development. Cas13, a novel RNA-targeting CRISPR effector protein, could bind and cleave complementary single-strand RNA, which has been employed for mRNA knockdown in mouse and human cells and RNA-virus interference in plants. Cas13 has not yet been reported to be used in pigs. In the current study, we explored the feasibility of CRISPR/Cas13d-mediated endogenous RNA knockdown in pigs. KDM5B, a histone demethylase of H3K4me3, was down-regulated at the transcriptional level by 50% with CRISPR/Cas13d in porcine fibroblast cells. Knockdown of KDM5B induced H3K4me3 expression and decreased the abundance of H3K27me3, H3K9me3, H3K4ac, H4K8ac, and H4K12ac. These changes affected cell proliferation and cell cycle. Furthermore, stable integration of the CRISPR/Cas13d system into the porcine genome resulted in the continuous expression of Cas13d and persistent knockdown of KDM5B. Finally, the RNA-targeting potential of Cas13d was further validated in porcine parthenogenetic embryos. By micro-injection of Cas13d mRNA and gRNA targeting KDM5B into porcine oocytes, the expression of KDM5B was down-regulated, the abundance of H3K4me3 increased as expected, and the expression of embryonic development-related genes was changed accordingly. These results indicate that CRISPR/Cas13d provides an easily programmable platform for spatiotemporal transcriptional manipulation in pigs.

The DDX23 Negatively Regulates Translation and Replication of Foot-and-Mouth Disease Virus and Is Degraded by 3C Proteinase

DEAD-box helicase 23 (DDX23) is a host nuclear helicase, which is a part of the spliceosomal complex and involved in pre-mRNA splicing. To investigate whether DDX23, an internal ribosomal entry sites transacting factor (ITAF) affects foot-and-mouth disease virus (FMDV) replication and translation through internal ribosome entry site (IRES)-dependent manner. For this, we utilized a pull-down assay, Western blotting, quantitative real-time PCR, confocal microscopy, overexpression and small interfering RNA knockdown, as well as the median tissue culture infective dose. Our findings showed that FMDV infection inhibited DDX23 expression and the overexpression of DDX23 reduced viral replication, however, CRISPR Cas9 knockout/small interfering RNA knockdown increased FMDV replication. FMDV IRES domain III and IV interacted with DDX23, whereas DDX23 interacted with FMDV 3C proteinase and significantly degraded. The enzymatic activity of FMDV 3C proteinase degraded DDX23, whereas FMDV degraded DDX23 via the lysosomal pathway. Additionally, IRES-driven translation was suppressed in DDX23-overexpressing cells, and was enhanced in DDX23 knocked down. Collectively, our results demonstrated that DDX23 negatively affects FMDV IRES-dependent translation, which could be a useful target for the design of antiviral drugs.

Dsi-RNA knockdown of genes regulated by Foxo reduces glycogen and lipid accumulations in diapausing Culex pipiens

Culex pipiens is a major carrier of the West Nile Virus, the leading cause of mosquito-borne disease in the continental United States. Cx. pipiens survive overwinter through diapause which is an important survival strategy that is under the control of insulin signaling and Foxo by regulating energy metabolism. Three homologous candidate genes, glycogen synthase (glys), atp-binding cassette transporter (atp), and low-density lipoprotein receptor chaperone (ldlr), that are under the regulation of Foxo transcription factor were identified in Cx. pipiens. To validate the gene functions, each candidate gene was silenced by injecting the target dsi-RNA to female Cx. pipiens during the early phase of diapause. The dsi-RNA injected diapause-destined female post-adult eclosion were fed for 7 days with 10% glucose containing 1% d-[13C6]glucose. The effects of dsi-RNA knockdown on glucose metabolism in intact mosquitoes were monitored using 13C solid-state NMR and ATR-FTIR. Our finding shows that the dsi-RNA knockdown of all three candidate genes suppressed glycogen and lipid biosyntheses resulting in inhibition of long-term carbon energy storage in diapausing females.

Vimentin filaments drive migratory persistence in polyploidal cancer cells

Polyploidal giant cancer cells (PGCCs) are multinucleated chemoresistant cancer cells found in heterogeneous solid tumors. Due in part to their apparent dormancy, the effect of PGCCs on cancer progression has remained largely unstudied. Recent studies have highlighted the critical role of PGCCs as aggressive and chemoresistant cancer cells, as well as their ability to undergo amitotic budding to escape dormancy. Our recent study demonstrated the unique biophysical properties of PGCCs, as well as their unusual migratory persistence. Here we unveil the critical function of vimentin intermediate filaments (VIFs) in maintaining the structural integrity of PGCCs and enhancing their migratory persistence. We performed in-depth single-cell analysis to examine the distribution of VIFs and their role in migratory persistence. We found that PGCCs rely heavily on their uniquely distributed and polarized VIF network to enhance their transition from a jammed to an unjammed state to allow for directional migration. Both the inhibition of VIFs with acrylamide and small interfering RNA knockdown of vimentin significantly decreased PGCC migration and resulted in a loss of PGCC volume. Because PGCCs rely on their VIF network to direct migration and to maintain their enlarged morphology, targeting vimentin or vimentin cross-linking proteins could provide a therapeutic approach to mitigate the impact of these chemoresistant cells in cancer progression and to improve patient outcomes with chemotherapy.