A versatile CRISPR-Cas13d platform for multiplexed transcriptomic regulation and metabolic engineering in primary human T cells.

CRISPR technologies have begun to revolutionize T cell therapies; however, conventional CRISPR-Cas9 genome-editing tools are limited in their safety, efficacy, and scope. To address these challenges, we developed multiplexed effector guide arrays (MEGA), a platform for programmable and scalable regulation of the T cell transcriptome using the RNA-guided, RNA-targeting activity of CRISPR-Cas13d. MEGA enables quantitative, reversible, and massively multiplexed gene knockdown in primary human T cells without targeting or cutting genomic DNA. Applying MEGA to a model of CAR T cell exhaustion, we robustly suppressed inhibitory receptor upregulation and uncovered paired regulators of T cell function through combinatorial CRISPR screening. We additionally implemented druggable regulation of MEGA to control CAR activation in a receptor-independent manner. Lastly, MEGA enabled multiplexed disruption of immunoregulatory metabolic pathways to enhance CAR T cell fitness and anti-tumor activity in vitro and in vivo. MEGA offers a versatile synthetic toolkit for applications in cancer immunotherapy and beyond.

Functional microRNA-targeting drug discovery by graph-based deep learning.

MicroRNAs are recognized as key drivers in many cancers but targeting them with small molecules remains a challenge. We present RiboStrike, a deep-learning framework that identifies small molecules against specific microRNAs. To demonstrate its capabilities, we applied it to microRNA-21 (miR-21), a known driver of breast cancer. To ensure selectivity toward miR-21, we performed counter-screens against miR-122 and DICER. Auxiliary models were used to evaluate toxicity and rank the candidates. Learning from various datasets, we screened a pool of nine million molecules and identified eight, three of which showed anti-miR-21 activity in both reporter assays and RNA sequencing experiments. Target selectivity of these compounds was assessed using microRNA profiling and RNA sequencing analysis. The top candidate was tested in a xenograft mouse model of breast cancer metastasis, demonstrating a significant reduction in lung metastases. These results demonstrate RiboStrike's ability to nominate compounds that target the activity of miRNAs in cancer.

Pre-twisting for improved genome modification and miRNA targeting.

Two reports by Dhuri et al. and Oyaghire et al., respectively, show that, through installing chiral centers at the backbone of the artificial nucleic acid, peptide nucleic acid (PNA), enhanced miRNA targeting and genome modification can be achieved, with important implications in fighting cancers and ß-thalassemia.

A New RNA-Dependent Cas12g Nuclease.

The development of RNA-targeting CRISPR-Cas systems represents a major step forward in the field of gene editing and regulation. RNA editing presents a viable alternative to genome editing in certain scenarios as it offers a reversible and manageable approach, reducing the likelihood of runaway mutant variants. One of the most promising applications is in the treatment of genetic disorders caused by mutations in RNA molecules. In this study, we investigate a previously undescribed Cas12g nuclease which was found in metagenomes from promising thermophilic microbial communities during the expedition to the Republic of North Ossetia-Alania in 2020. The method outlined in this study can be applied to other Cas orthologs and variants, leading to a better understanding of the CRISPR-Cas system and its enzymatic activities. The cis-cleavage activity of the new type V-G Cas effector was indicated by in vitro RNA cleavage experiments. While CRISPR-Cas systems are known for their high specificity, there is still a risk of unintended cleavage of nontargeted RNA molecules. Ultimately, the search for new genome editing tools and the study of their properties will remove barriers to research in this area. With continued research and development, we may be able to unlock their full potential.

Antibody-drug conjugates: Recent advances in payloads.

Antibody‒drug conjugates (ADCs), which combine the advantages of monoclonal antibodies with precise targeting and payloads with efficient killing, show great clinical therapeutic value. The ADCs' payloads play a key role in determining the efficacy of ADC drugs and thus have attracted great attention in the field. An ideal ADC payload should possess sufficient toxicity, low immunogenicity, high stability, and modifiable functional groups. Common ADC payloads include tubulin inhibitors and DNA damaging agents, with tubulin inhibitors accounting for more than half of the ADC drugs in clinical development. However, due to clinical limitations of traditional ADC payloads, such as inadequate efficacy and the development of acquired drug resistance, novel highly efficient payloads with diverse targets and reduced side effects are being developed. This perspective summarizes the recent research advances of traditional and novel ADC payloads with main focuses on the structure-activity relationship studies, co-crystal structures, and designing strategies, and further discusses the future research directions of ADC payloads. This review also aims to provide valuable references and future directions for the development of novel ADC payloads that will have high efficacy, low toxicity, adequate stability, and abilities to overcome drug resistance.

Cas13a-based multiplex RNA targeting for potato virus Y.

MAIN CONCLUSION: The Cas13a-based multiplex RNA targeting system can be engineered to confer resistance to RNA viruses, whereas the number and expression levels of gRNAs have no significant effect on viral interference. The CRISPR-Cas systems provide adaptive immunity to bacterial and archaeal species against invading phages and foreign plasmids. The class 2 type VI CRISPR/Cas effector Cas13a has been harnessed to confer the protection against RNA viruses in diverse eukaryotic species. However, whether the number and expression levels of guide RNAs (gRNAs) have effects on the efficiency of RNA virus inhibition is unknown. Here, we repurpose CRISPR/Cas13a in combination with an endogenous tRNA-processing system (polycistronic tRNA-gRNA) to target four genes of potato virus Y (PVY) with varying expression levels. We expressed Cas13a and four different gRNAs in potato lines, and the transgenic plants expressing multiple gRNAs displayed similar suppression of PVY accumulation and reduced disease symptoms as those expressing a single gRNA. Moreover, PTG/Cas13a-transformed plants with different expression levels of multiple gRNAs displayed similar resistance to PVY strains. Collectively, this study suggests that the Cas13a-based multiplex RNA targeting system can be utilized to engineer resistance to RNA viruses in plants, whereas the number and expression levels of gRNAs have no significant effect on CRISPR/Cas13a-mediated viral interference in plants.

Research progress in mRNA drug modification and delivery systems. / 信使RNAè¯ç‰©ä¿®é¥°åŠå ¶é€’é€ç³»ç»Ÿç ”ç©¶è¿›å±•.

Messenger RNA (mRNA) has shown tremendous potential in disease prevention and therapy. The clinical application requires mRNA with enhanced stability and high translation efficiency, ensuring it not to be degraded by nucleases and targeting to specific tissues and cells. mRNA immunogenicity can be reduced by nucleotide modification, and translation efficiency can be enhanced by codon optimization. The 5´ capping structure and 3´ poly A increase mRNA stability, and the addition of 5' and 3' non-translational regions regulate mRNA translation initiation and protein production. Nanoparticle delivery system protects mRNA from degradation by ubiquitous nucleases, enhances mRNA concentration in circulation and assists it cytoplasmic entrance for the purpose of treatment and prevention. Here, we review the recent advances of mRNA technology, discuss the methods and principles to enhance mRNA stability and translation efficiency; summarize the requirements involved in designing mRNA delivery systems with the potential for industrial translation and biomedical application. Furthermore, we provide insights into future directions of mRNA therapeutics to meet the needs for personalized precision medicine.

Progress of CRISPR-based programmable RNA manipulation and detection.

Prokaryotic clustered regularly interspaced short palindromic repeats and CRISPR associated (CRISPR-Cas) systems provide adaptive immunity by using RNA-guided endonucleases to recognize and eliminate invading foreign nucleic acids. Type II Cas9, type V Cas12, type VI Cas13, and type III Csm/Cmr complexes have been well characterized and developed as programmable platforms for selectively targeting and manipulating RNA molecules of interest in prokaryotic and eukaryotic cells. These Cas effectors exhibit remarkable diversity of ribonucleoprotein (RNP) composition, target recognition and cleavage mechanisms, and self discrimination mechanisms, which are leveraged for various RNA targeting applications. Here, we summarize the current understanding of mechanistic and functional characteristics of these Cas effectors, give an overview on RNA detection and manipulation toolbox established so far including knockdown, editing, imaging, modification, and mapping RNA-protein interactions, and discuss the future directions for CRISPR-based RNA targeting tools. This article is categorized under: RNA Methods > RNA Analyses in Cells RNA Processing > RNA Editing and Modification RNA Interactions with Proteins and Other Molecules > Protein-RNA Interactions: Functional Implications.

Investigating the NRAS 5' UTR as a target for small molecules.

Neuroblastoma RAS (NRAS) is an oncogene that is deregulated and highly mutated in cancers including melanomas and acute myeloid leukemias. The 5' untranslated region (UTR) (5' UTR) of the NRAS mRNA contains a G-quadruplex (G4) that regulates translation. Here we report a novel class of small molecule that binds to the G4 structure located in the 5' UTR of the NRAS mRNA. We used a small molecule microarray screen to identify molecules that selectively bind to the NRAS-G4 with submicromolar affinity. One compound inhibits the translation of NRAS in vitro but showed only moderate effects on the NRAS levels in cellulo. Rapid Amplification of cDNA Ends and RT-PCR analysis revealed that the predominant NRAS transcript does not possess the G4 structure. Thus, although NRAS transcripts lack a G4 in many cell lines the concept of targeting folded regions within 5' UTRs to control translation remains a highly attractive strategy.

The effects of length and sequence of gRNA on Cas13b and Cas13d activity in vitro and in vivo.

Cas13 are the only CRISPR/Cas systems found so far, which target RNA strand while preserving chromosomal integrity. Cas13b or Cas13d cleaves RNA by the crRNA guidance. However, the effect of the characteristics of the spacer sequences, such as the length and sequence preference, on the activity of Cas13b and Cas13d remains unclear. Our study shows that neither Cas13b nor Cas13d has a particular preference for the sequence composition of gRNA, including the sequence of crRNA and its flanking sites on target RNA. However, the crRNA, complementary to the middle part of the target RNA, seems to show higher cleavage efficiency for both Cas13b and Cas13d. As for the length of crRNAs, the most appropriate crRNA length for Cas13b is 22-25 nt and crRNA as short as 15 nt is still functional. Whereas, Cas13d requires longer crRNA, and 22-30 nt crRNA can achieve good effect. Both Cas13b and Cas13d show the ability to process precursor crRNAs. Our study suggests that Cas13b may have a stronger precursor processing ability than Cas13d. There are few in vivo studies on the application of Cas13b or Cas13d in mammals. With the methods of transgenic mice and hydrodynamic injection via tail vein, our study showed that both of them had high knock-down efficiency against target RNA in vivo. These results indicate that Cas13b and Cas13d have great potential for in vivo RNA operation and disease treatment without damaging genomic DNA.

RNA-Dependent RNA Targeting by CRISPR-Cas Systems: Characterizations and Applications.

Genome editing technologies that are currently available and described have a fundamental impact on the development of molecular biology and medicine, industrial and agricultural biotechnology and other fields. However, genome editing based on detection and manipulation of the targeted RNA is a promising alternative to control the gene expression at the spatiotemporal transcriptomic level without complete elimination. The innovative CRISPR-Cas RNA-targeting systems changed the conception of biosensing systems and also allowed the RNA effectors to be used in various applications; for example, genomic editing, effective virus diagnostic tools, biomarkers, transcription regulations. In this review, we discussed the current state-of-the-art of specific CRISPR-Cas systems known to bind and cleave RNA substrates and summarized potential applications of the versatile RNA-targeting systems.

Targeting RNA with small molecules: lessons learned from Xist RNA.

Although more than 98% of the human genome is noncoding, nearly all drugs on the market target one of about 700 disease-related proteins. However, an increasing number of diseases are now being attributed to noncoding RNA and the ability to target them would vastly expand the chemical space for drug development. We recently devised a screening strategy based upon affinity-selection mass spectrometry and succeeded in identifying bioactive compounds for the noncoding RNA prototype, Xist. One such compound, termed X1, has drug-like properties and binds specifically to the RepA motif of Xist in vitro and in vivo. Small-angle X-ray scattering analysis reveals that X1 changes the conformation of RepA in solution, thereby explaining the displacement of cognate interacting protein factors (PRC2 and SPEN) and inhibition of X-chromosome inactivation. In this Perspective, we discuss lessons learned from these proof-of-concept experiments and suggest that RNA can be systematically targeted by drug-like compounds to disrupt RNA structure and function.

A New Method for Programmable RNA Editing Using CRISPR Effector Cas13X.1.

Type VI CRISPR-Cas13 is the only CRISPR system that can bind and cleave RNA without DNase activity. We used the newly discovered, smaller Cas13X.1 protein to construct an editing system in mammalian cells, aiming to break the delivery restrictions of CRISPR-Cas13 system in vivo and promote the application of Cas13X system in clinical therapy. We employed exogenous fluorescence reporter gene mCherry and endogenous gene transketolase (TKT) closely related to cancer cell metabolism as target genes to evaluate the Cas13X.1 system. The recombinant plasmids targeting exogenous gene mCherry and endogenous gene TKT were constructed based on Cas13X.1 backbone plasmid. The editing efficiency, protein expression level, downstream gene transcript level and safety of Cas13X.1 system were evaluated. Both TKT transcripts of endogenous genes and mCherry transcripts of exogenous genes were significantly degraded by Cas13X.1 system with a knockdown efficiency up to 50%. At the same time, Cas13X.1 down-regulated the expression of the corresponding protein level in the editing of transcripts. In addition, the transcripts of key metabolic enzymes related to TKT were also down-regulated synchronously, suggesting that the degradation of TKT transcripts by Cas13X.1 system affected the main metabolic pathways related to TKT. The morphology, RNA integrity and apoptosis of cells loaded with Cas13X.1 system were not affected. The Cas13X.1 system we constructed had strong RNA knockdown ability in mammalian cells with low cellular toxicity. Compared with other CRISPR-Cas13 systems, Cas13X.1 system with smaller molecular weight has more advantages in vivo delivery. The Cas13X.1 system targeting TKT transcripts also provides an alternative method for the study of anti-cancer therapy.

RNA-Targeting Carbon Dots for Live-Cell Imaging of Granule Dynamics.

It is significant to monitor the different RNA granules dynamics and phase separation process inside cells under various stresses, for example, oxidative stress. The current small-molecule RNA probes work well only in fixed cells and usually encounter problems such as insufficient stability and biocompatibility, and thus a specific RNA-targeting fluorescent nanoprobe that can be used in the living cells is urgently desired. Here, the de novo design and microwave-assisted synthesis of a novel RNA-targeting, red-emissive carbon dots (named as M-CDs) are reported by choosing neutral red and levofloxacin as precursors. The as-synthesized M-CDs is water-soluble with a high fluorescence quantum yield of 22.83% and can selectively bind to RNA resulting in an enhanced red fluorescence. More interestingly, such an RNA-targeting, red-emissive M-CDs can be fast internalized into cells within 5 s and thus used for real-time imaging the dynamic process of intracellular stress granules under oxidative stress, revealing some characteristics of granules that have not been identified by previously reported RNA and protein biomarkers. This research paves a new pathway for visualizing bulk RNA dynamics and studying phase-separation behaviors in living cells by rational design of the fluorescent carbon dots in terms of structure and functionality.

Programmable macromolecule-based RNA-targeting therapies to treat human neurological disorders.

Disruptions in RNA processing play critical roles in the pathogenesis of neurological diseases. In this Perspective, we discuss recent progress in the development of RNA-targeting therapeutic modalities. We focus on progress, limitations, and opportunities in a new generation of therapies engineered from RNA binding proteins and other endogenous RNA regulatory macromolecules to treat human neurological disorders.

Characterization of the structure and control of the blood-nerve barrier identifies avenues for therapeutic delivery.

The blood barriers of the nervous system protect neural environments but can hinder therapeutic accessibility. The blood-brain barrier (BBB) is well characterized, consisting of endothelial cells with specialized tight junctions and low levels of transcytosis, properties conferred by contacting pericytes and astrocytes. In contrast, the blood-nerve barrier (BNB) of the peripheral nervous system is poorly defined. Here, we characterize the structure of the mammalian BNB, identify the processes that confer barrier function, and demonstrate how the barrier can be opened in response to injury. The homeostatic BNB is leakier than the BBB, which we show is due to higher levels of transcytosis. However, the barrier is reinforced by macrophages that specifically engulf leaked materials, identifying a role for resident macrophages as an important component of the BNB. Finally, we demonstrate the exploitation of these processes to effectively deliver RNA-targeting therapeutics to peripheral nerves, indicating new treatment approaches for nervous system pathologies.

RNA interference (RNAi)-based therapeutics for treatment of rare neurologic diseases.

Advances in genome sequencing have greatly facilitated the identification of genomic variants underlying rare neurodevelopmental and neurodegenerative disorders. Understanding the fundamental causes of rare monogenic disorders has made gene therapy a possible treatment approach for these conditions. RNA interference (RNAi) technologies such as small interfering RNA (siRNA), microRNA (miRNA), and short hairpin RNA (shRNA), and other oligonucleotide-based modalities such as antisense oligonucleotides (ASOs) are being developed as potential therapeutic approaches for manipulating expression of the genes that cause a variety of neurological diseases. Here, we offer a brief review of the mechanism of action of these RNAi approaches; provide deeper discussion of the advantages, challenges, and specific considerations related to the development of RNAi therapeutics for neurological disease; and highlight examples of rare neurological diseases for which RNAi therapeutics hold great promise.

CRISPR-free, programmable RNA pseudouridylation to suppress premature termination codons.

Nonsense mutations, accounting for >20% of disease-associated mutations, lead to premature translation termination. Replacing uridine with pseudouridine in stop codons suppresses translation termination, which could be harnessed to mediate readthrough of premature termination codons (PTCs). Here, we present RESTART, a programmable RNA base editor, to revert PTC-induced translation termination in mammalian cells. RESTART utilizes an engineered guide snoRNA (gsnoRNA) and the endogenous H/ACA box snoRNP machinery to achieve precise pseudouridylation. We also identified and optimized gsnoRNA scaffolds to increase the editing efficiency. Unexpectedly, we found that a minor isoform of pseudouridine synthase DKC1, lacking a C-terminal nuclear localization signal, greatly improved the PTC-readthrough efficiency. Although RESTART induced restricted off-target pseudouridylation, they did not change the coding information nor the expression level of off-targets. Finally, RESTART enables robust pseudouridylation in primary cells and achieves functional PTC readthrough in disease-relevant contexts. Collectively, RESTART is a promising RNA-editing tool for research and therapeutics.

Research progress in mRNA drug modification and delivery systems / 浙江大学学报·医学版

Messenger RNA (mRNA) has shown tremendous potential in disease prevention and therapy. The clinical application requires mRNA with enhanced stability and high translation efficiency, ensuring it not to be degraded by nucleases and targeting to specific tissues and cells. mRNA immunogenicity can be reduced by nucleotide modification, and translation efficiency can be enhanced by codon optimization. The 5´ capping structure and 3´ poly A increase mRNA stability, and the addition of 5' and 3' non-translational regions regulate mRNA translation initiation and protein production. Nanoparticle delivery system protects mRNA from degradation by ubiquitous nucleases, enhances mRNA concentration in circulation and assists it cytoplasmic entrance for the purpose of treatment and prevention. Here, we review the recent advances of mRNA technology, discuss the methods and principles to enhance mRNA stability and translation efficiency; summarize the requirements involved in designing mRNA delivery systems with the potential for industrial translation and biomedical application. Furthermore, we provide insights into future directions of mRNA therapeutics to meet the needs for personalized precision medicine.

Antibody-drug conjugates: Recent advances in payloads

Antibody‒drug conjugates (ADCs), which combine the advantages of monoclonal antibodies with precise targeting and payloads with efficient killing, show great clinical therapeutic value. The ADCs' payloads play a key role in determining the efficacy of ADC drugs and thus have attracted great attention in the field. An ideal ADC payload should possess sufficient toxicity, low immunogenicity, high stability, and modifiable functional groups. Common ADC payloads include tubulin inhibitors and DNA damaging agents, with tubulin inhibitors accounting for more than half of the ADC drugs in clinical development. However, due to clinical limitations of traditional ADC payloads, such as inadequate efficacy and the development of acquired drug resistance, novel highly efficient payloads with diverse targets and reduced side effects are being developed. This perspective summarizes the recent research advances of traditional and novel ADC payloads with main focuses on the structure-activity relationship studies, co-crystal structures, and designing strategies, and further discusses the future research directions of ADC payloads. This review also aims to provide valuable references and future directions for the development of novel ADC payloads that will have high efficacy, low toxicity, adequate stability, and abilities to overcome drug resistance.