Rewriting endogenous human transcripts with dual CRISPR-guided 3' trans-splicing.

Since the recombinant plasmid was first introduced into E.coli by Cohen et al. [1] in 1973, genetic engineering (modification) has been developing rapidly. Genetic modifications in vivo or in vitro, which have an effect on the expression of specific proteins, such as channel, receptor, enzyme, and signals, are actively applied in the researches that highlight the relationship between the effects of anesthetics and functions of specific proteins [2-5]. The loss-of-function studies that control the function of proteins, are very important in clarifying the mechanism of action of anesthetics. To achieve this, post-translational proteins are directly inhibited by pharmacological inhibitors [6,7], or are indirectly suppressed by transforming DNA construct via mutation or recombination, which is known as transgenic knockout technique [8]. After the creation of knockout mice by Capecchi et al. in 1989, transgenic knockout techniques are not only available in vitro, but are also available in vivo animal studies (In 2007, Capecchi MR won a nobel prize in appreciation for creating transgenic knockout mice). Although the gene knockout technique has several advantages over conventional laboratory techniques, including a long-lasting and a definitive specific gene-silencing effect, it is not easy to get a genetic engineering technique and initially it is expensive to purchase a gene knockout animal. After the discovery of RNA interference (RNAi) that is induced by short sequence-specific double stranded RNA (dsRNA) ranging in size from 20 to 25 nucleotides in length, and that targets the complementary sites on mRNA, exogenous synthetic small interfering RNA (siRNA) could be used to induce post-transcriptional gene silencing and that was first applied in mammalian cells in 2001 [9]. Because well-designed siRNA can induce suppression of any gene through forming the RNA- induced silencing complex (RISC) in the cytoplasm, the research for therapeutic application of siRNA against cancer, viral infection, and neurodegenerative disease are now being performed and clinical trials are also being carried out [10,11]. Moreover, if methods for effective in vivo delivery of siRNAs into the target tissue or organ are developed, anesthesiologists could easily achieve control over the tissue or organ-specific expression of individual target proteins used in the research. It is known that systemically or locally delivered siRNA induces a temporary gene expression knockdown effect by up to 90% from 48 hours to 3 weeks in animal experiments for eyes, brain, spinal cord, lungs, subcutaneous tissue, vagina, skin, isolated tumor, heart et al. [10-12]. But systemically or locally administered siRNA is easily degraded during the phase of delivery to the target cells by endogenous enzymes or phagocytosis, and its permeation through the negatively charged hydrophobic cellular membranes is difficult because of its high molecular weight (~13 kDa) and its too negatively charged character. Also, the cytoplasmic concentration of transfected siRNA is reduced as cell divisions progress and, cell types and pericellular factors influencing cell division may influence the gene silencing effects over time [13]. In general, the small size of the siRNA is considered not to provoke cytosolic dsRNA-mediated interferon response. But, it is reported that transfected siRNA is linked to the production of interferon and interleukin in some cell types (plasmacytoid dentritic cells, macrophages), that may be related to immunotoxicity. And, it is also known that a siRNA could degrade not only the targeted mRNAs but also the unintended mRNAs with sequence homology with the siRNA, which is called the off-target effect. As a result, there is a chance of inaccurate results in the experiments undertaken using the suppression of a specific protein and even for assessing the cytotoxicity. To correct such shortcomings, it is recommended that the administered doses of siRNA should be reduced while constructing the design of experiments for successful delivery of siRNA into a target cell or tissue. And, the nucleotide modifications of siRNA can be applied in order to prevent the 'off-target effect' [13]. Ki et al. [14] in this month's journal found the lowest concentration of siRNA against a specific protein that could not only induce effective transfection of siRNA but also show maximum RNAi-mediated gene silencing while not demonstrating cytotoxicity, both in the cultured astrocytes and microglial cells [14]. Identifying the lowest effective concentration of siRNA is desirable for minimizing the adverse reactions and also for improving the cost-effectiveness for researchers who are always confronted with the problem of lack of financial support for experiments. Although Ki et al. [14] have clarified that each cell type has its own characteristic for determining the lowest concentration of siRNA against a specific protein at which the targeted mRNA is effectively silenced (5 nM for astrocytes, 20 nM for microglial cells), it was not determined whether that concentration of siRNA in each cell is related to the 'off-target effect' (knockdown of untargeted proteins). Tschuch et al. [15] reported that 5 nM siRNA against green fluorescent protein (GFP), which was the protein used in the study by Ki et al. [14], can suppress the expression of the other unintended proteins (CYLD and SOAT) in HeLa and HEK cells. This fact implies that untargeted proteins may also be suppressed by 20 nM siRNA against GFP, the lowest concentration suppressing the target gene expression both in the astrocytes and microglial cells in the study by Ki et al. [14], and thereby the accuracy of results of the study may be adversely affected. Thus before performing research using the procedure for siRNA silencing of expression of a specific protein, it is necessary not only to find out the lowest concentration at which a target protein could be controlled, but also to investigate the concentration of siRNA at which cellular apoptosis and expression of stress-response proteins are not triggered [16]. Ki et al. [14] tried to estimate the degree of cytotoxicity at the measured concentration of siRNA using the MTT assay, by which cell viability can be assessed. At present, control of protein expression by siRNA-induced transcriptional gene silencing is on the rise in the field of laboratory research. But, siRNA can provoke 'off-target effect' and immunomodulation, and the time course of siRNA-induced gene silencing is still unclear. So, results of the study may be adversely affected. Further research is needed to confirm the accuracy of results of the RNA interference experiments using siRNA.

Novel Engineered Programmable Systems for ADAR-Mediated RNA Editing

RNA editing, particularly A-to-I RNA editing, has been shown to play an essential role in mammalian embryonic development and tissue homeostasis, and is implicated in the pathogenesis of many diseases including skin pigmentation disorder, autoimmune and inflammatory tissue injury, neuron degeneration, and various malignancies. A-to-I RNA editing is carried out by a small group of enzymes, the adenosine deaminase acting on RNAs (ADARs). Only three members of this protein family, ADAR1–3, exist in mammalian cells. ADAR3 is a catalytically null enzyme and the most significant function of ADAR2 was found to be in editing on the neuron receptor GluR-B mRNA. ADAR1, however, has been shown to play more significant roles in biological and pathological conditions. Although there remains much that is not known about how ADAR1 regulates cellular function, recent findings point to regulation of the innate immune response as an important function of ADAR1. Without appropriate RNA editing by ADAR1, endogenous RNA transcripts stimulate cytosolic RNA sensing receptors and therefore activate the IFN-inducing signaling pathways. Overactivation of innate immune pathways can lead to tissue injury and dysfunction. However, obvious gaps in our knowledge persist as to how ADAR1 regulates innate immune responses through RNA editing. Here, we review critical findings from ADAR1 mechanistic studies focusing on its regulatory function in innate immune responses and identify some of the important unanswered questions in the field.

Adenosine-to-inosine (A-to-I) editing is a highly prevalent posttranscriptional modification of RNA, mediated by ADAR (adenosine deaminase acting on RNA) enzymes. In addition to RNA editing, additional functions have been proposed for ADAR1. To determine the specific role of RNA editing by ADAR1, we generated mice with an editing-deficient knock-in mutation (Adar1(E861A), where E861A denotes Glu(861)→Ala(861)). Adar1(E861A/E861A) embryos died at ~E13.5 (embryonic day 13.5), with activated interferon and double-stranded RNA (dsRNA)-sensing pathways. Genome-wide analysis of the in vivo substrates of ADAR1 identified clustered hyperediting within long dsRNA stem loops within 3' untranslated regions of endogenous transcripts. Finally, embryonic death and phenotypes of Adar1(E861A/E861A) were rescued by concurrent deletion of the cytosolic sensor of dsRNA, MDA5. A-to-I editing of endogenous dsRNA is the essential function of ADAR1, preventing the activation of the cytosolic dsRNA response by endogenous transcripts.

BackgroundA-to-I RNA editing is a co−/post-transcriptional modification catalyzed by ADAR enzymes, that deaminates Adenosines (A) into Inosines (I). Most of known editing events are located within inverted ALU repeats, but they also occur in coding sequences and may alter the function of encoded proteins. RNA editing contributes to generate transcriptomic diversity and it is found altered in cancer, autoimmune and neurological disorders. Emerging evidences indicate that editing process could be influenced by genetic variations, biological and environmental variables.ResultsWe analyzed RNA editing levels in human blood using RNA-seq data from 459 healthy individuals and identified 2079 sites consistently edited in this tissue. As expected, analysis of gene expression revealed that ADAR is the major contributor to editing on these sites, explaining ~ 13% of observed variability. After removing ADAR effect, we found significant associations for 1122 genes, mainly involved in RNA processing. These genes were significantly enriched in genes encoding proteins interacting with ADARs, including 276 potential ADARs interactors and 9 ADARs direct partners. In addition, our analysis revealed several factors potentially influencing RNA editing in blood, including cell composition, age, Body Mass Index, smoke and alcohol consumption. Finally, we identified genetic loci associated with editing levels, including known ADAR eQTLs and a small region on chromosome 7, containing LOC730338, a lincRNA gene that appears to modulate ADARs mRNA expression.ConclusionsOur data provides a detailed picture of the most relevant RNA editing events and their variability in human blood, giving interesting insights on potential mechanisms behind this post-transcriptional modification and its regulation in this tissue.

<div>Abstract<p>RNA editing modifies single nucleotides of RNAs, regulating primary protein structure and protein abundance. In recent years, the diversity of proteins and complexity of gene regulation associated with RNA editing dysregulation has been increasingly appreciated in oncology. Large-scale shifts in editing have been observed in bulk tumors across various cancer types. However, RNA editing in single cells and individual cell types within tumors has not been explored. By profiling editing in single cells from lung adenocarcinoma biopsies, we found that the increased editing trend of bulk lung tumors was unique to cancer cells. Elevated editing levels were observed in cancer cells resistant to targeted therapy, and editing sites associated with drug response were enriched. Consistent with the regulation of antiviral pathways by RNA editing, higher editing levels in cancer cells were associated with reduced antitumor innate immune response, especially levels of natural killer cell infiltration. In addition, the level of RNA editing in cancer cells was positively associated with somatic point mutation burden. This observation motivated the definition of a new metric, RNA editing load, reflecting the amount of RNA mutations created by RNA editing. Importantly, in lung cancer, RNA editing load was a stronger predictor of patient survival than DNA mutations. This study provides the first single cell dissection of editing in cancer and highlights the significance of RNA editing load in cancer prognosis.</p>Significance:<p>RNA editing analysis in single lung adenocarcinoma cells uncovers RNA mutations that correlate with tumor mutation burden and cancer innate immunity and reveals the amount of RNA mutations that strongly predicts patient survival.</p><p><i><a href="https://aacrjournals.org/cancerres/article/doi/10.1158/0008-5472.CAN-22-3537" target="_blank">See related commentary by Luo and Liang, p. 351</a></i></p></div>

CRISPR technology provides unprecedented precision for molecular imaging and target detection at the cellular level. However, interested researchers inevitably encounter challenges, including weak labeling signals, low delivery efficiency, and off-target effects when establishing application frameworks. Therefore, discussing signal labeling strategies and delivery systems is crucial to further improving the performance of CRISPR-based live cell biosensing. In this review, we first focus on signal labeling strategies for CRISPR-based cellular imaging, including fluorescent protein fusion, fluorescent protein recruitment, RNA modification and hybridization, and irrelevant signal reporters. Depending on the effector proteins and application scenarios, selecting the appropriate signal labeling method can help improve imaging sensitivity and signal intensity. Second, we summarize materials that can be used for CRISPR intracellular delivery, such as nanoparticles, nanosheets, and other nanomaterials. Some nanomaterials have been shown to further enhance the activity of effector proteins, in addition to facilitating the cellular entry of active components. Furthermore, we discuss the challenges and future directions of CRISPR imaging technologies. By integrating multidisciplinary innovations, the CRISPR-based live cell biosensor holds promise as a next-generation visualization tool for life science research and precision medicine.

Preclinical mechanistic studies have pointed towards RNA interference-mediated off-target effects as a major driver of hepatotoxicity for GalNAc–siRNA conjugates. Here, we demonstrate that a single glycol nucleic acid or 2′–5′-RNA modification can substantially reduce small interfering RNA (siRNA) seed-mediated binding to off-target transcripts while maintaining on-target activity. In siRNAs with established hepatotoxicity driven by off-target effects, these novel designs with seed-pairing destabilization, termed enhanced stabilization chemistry plus (ESC+), demonstrated a substantially improved therapeutic window in rats. In contrast, siRNAs thermally destabilized to a similar extent by the incorporation of multiple DNA nucleotides in the seed region showed little to no improvement in rat safety suggesting that factors in addition to global thermodynamics play a role in off-target mitigation. We utilized the ESC+ strategy to improve the safety of ALN-HBV, which exhibited dose-dependent, transient and asymptomatic alanine aminotransferase elevations in healthy volunteers. The redesigned ALN-HBV02 (VIR-2218) showed improved specificity with comparable on-target activity and the program was reintroduced into clinical development.

Modified siRNA Structure With a Single Nucleotide Bulge Overcomes Conventional siRNA-mediated Off-target Silencing

Exploiting Endogenous Micro-RNAs to Avoid off-Target Transgene Expression

In recent years, RNA trans-splicing has emerged as a suitable RNA editing tool for the specific replacement of mutated gene regions at the pre-mRNA level. Although the technology has been successfully applied for the restoration of protein function in various genetic diseases, a higher trans-splicing efficiency is still desired to facilitate its clinical application. Here, we describe a modified, easily applicable, fluorescence-based screening system for the generation and analysis of antisense molecules specifically capable of improving the RNA reprogramming efficiency of a selected KRT14-specific RNA trans-splicing molecule. Using this screening procedure, we identified several antisense RNAs and short rationally designed oligonucleotides, which are able to increase the trans-splicing efficiency. Thus, we assume that besides the RNA trans-splicing molecule, short antisense molecules can act as splicing modulators, thereby increasing the trans-splicing efficiency to a level that may be sufficient to overcome the effects of certain genetic predispositions, particularly those associated with dominantly inherited diseases.

### Widespread RNA and DNA Sequence Differences in the Human Transcriptome Li et al Science . 2011;333:53–58. New genomic studies show that the transfer of genetic information from DNA to RNA within humans is not very faithful. If true, frequent, or purposeful, then the implications for human health and disease are significant, but the burden of proof required is significant. Commentary: Molecular biology and genetics has a “central dogma.” First postulated by Francis Crick1,2 in 1958, shortly after the discovery of the double helix, it states that the molecular transfer or flow of genetic information occurs only as DNA→RNA→ protein. This argument is intrinsic to our understanding of genotypes, phenotypes, and human disease. The central “dogma,” a word whose precise meaning Crick acknowledged he did not precisely know3 and that could have been given the less glamorous moniker “hypothesis,” is frequently misunderstood as also implying high accuracy or even inflexibility.2 We now know that genetic information flow can be sometimes corrupted and can also occur in other directions in particular biological systems. A recent study by Li and colleagues,4 from the laboratory of Vivian Cheung at the University of Pennsylvania, now demonstrates apparent widespread DNA→RNA sequence differences in humans. The experiments they conducted involved sequencing reverse-transcribed RNA from cultured B cells isolated from 27 different humans and comparing them with their host genome sequences. Surprisingly, after appropriate quality controls, they discovered numerous base positions at which the RNA sequence was different from the DNA sequence in multiple molecules, a phenomenon they refer to as RDD (RNA-DNA difference). Specifically, they identified 28766 RDDs in the 27 individuals at 10210 exonic sites within 4741 genes. Each individual had high-quality RNA-based sequence data at 9.8 million sites: so, RDD was detected at a rate of 1.1×10−4 per exonic site (base) across human genes. Interestingly, they observed all 12 types …

AMPA Receptor Subunit-Specific Regulation by a Distinct Family of Type II TARPs

Genetically modified mice have advanced our knowledge on platelets in hemostasis and beyond tremendously. However, mouse models harbor certain limitations, including availability of platelet specific transgenic strains, and off-target effects on other cell types. Transfusion of genetically modified platelets into thrombocytopenic mice circumvents these problems. Additionally, ex vivo treatment of platelets prior to transfusion eliminates putative side effects on other cell types. Thrombocytopenia is commonly induced by administration of anti-platelet antibodies, which opsonize platelets to cause rapid clearance. However, antibodies do not differentiate between endogenous or exogenous platelets, impeding transfusion efficacy. In contrast, genetic depletion with the inducible diphtheria toxin receptor (iDTR) system induces thrombocytopenia via megakaryocyte ablation without direct effects on circulating platelets. We compared the iDTR system with antibody-based depletion methods regarding their utility in platelet transfusion experiments, outlining advantages and disadvantages of both approaches. Antibodies led to thrombocytopenia within two hours and allowed the dose-dependent adjustment of the platelet count. The iDTR model caused complete thrombocytopenia within four days, which could be sustained for up to 11 days. Neither platelet depletion approach caused platelet activation. Only the iDTR model allowed efficient platelet transfusion by keeping endogenous platelet levels low and maintaining exogenous platelet levels over longer time periods, thus providing clear advantages over antibody-based methods. Transfused platelets were fully functional in vivo, and our model allowed examination of transgenic platelets. Using donor platelets from already available genetically modified mice or ex vivo treated platelets, may decrease the necessity of platelet-specific mouse strains, diminishing off-target effects and thereby reducing animal numbers.

Compelling murine studies demonstrate that adenosine-to-inosine (A-to-I) RNA editing mediated by adenosine deaminase associated with RNA1 (ADAR1) is vital for both fetal and adult hematopoiesis. While genetic ablation of ADAR1 leads to murine embryonic lethality due to severe defects in erythropoiesis, conditional deletion in the hematopoietic system impairs maintenance indicative of cell type and context specific roles for ADAR1 in cell fate specification and self-renewal. By regulating mRNA and microRNA (miRNA) stability, ADAR1 exhibit wide-ranging effects on embryonic development and stem cell regulation. We have previously shown that inflammation-responsive ADAR1 plays important roles in both stem cell differentiation and self-renewal in CML (chronic myeloid leukemia) disease progression. Here, we describe a novel function of ADAR1 in cell cycle regulation of normal hematopoietic stem cell and progenitors (HSPC) by regulation of miRNA biogenesis. Our results demonstrated that ADAR1 induces G0 to G1 phase transition in normal cord blood HSPCs, as demonstrated by elevated expression of Ki67, reduced DiR signal, and enhanced in vivo cord blood engraftment. Cell cycle qRT-qPCR microarray of 84 cell cycle transcripts and whole transcriptome RNA-sequencing analysis of KEGG cell cycle pathway indicate that several cell cycle genes are differentially expression upon overexpression of ADAR1 WT or an A-to-I editing deficient ADAR1 mutant (ADAR1E912A). We previously demonstrated that impaired biogenesis of let-7 miRNAs by ADAR1 WT induces enhanced self-renewal in cord blood CD34+ HSPCs. To determine the miRNA targets of ADAR1-mediated RNA editing, we performed miRNome miScript PCR array of 1008 miRNA candidates in cord blood CD34+ HSPCs overexpressing ADAR1 WT or ADAR1E912A. Total of 263 miRNAs were differentially expressed (142 upregulated and 121 downregulated) by comparing ADAR1 WT to the backbone control. Interestingly, ADAR1E912A mutant also exhibit A-to-I editing independent regulation of miRNAs (307 upregulated and 59 downregulated). We found that the expression of miR-26a-5p, a miRNA frequently downregulated in leukemia, is inhibited by ADAR1-mediated RNA editing. ADAR1 directly binds and edits the DROSHA cleavage site of primary miR-26a transcript, thereby prevent miR26a-5p maturation. Moreover, lentiviral expression of mature miR26-5p reverses the effect of ADAR1 WT, including enhanced CDKN1A expression, inhibition of cord blood proliferation in vivo, as well as reduced HSC self-renewal as measured by colony-formation assay. Our finding suggests carefully regulated A-to-I editing by ADAR1 is essential for the maintenance of proper cell proliferation in HSC. For future study, it will be interesting to investigate if the elevated expression of ADAR1 in CML BC LSC contributes to false regulation of cell cycle that leads to the expansion of malignant leukemia stem cells. Citation Format: Qingfei Jiang, Maria Anna Zipeto, Nathan Delos Santos, Sheldon Morris, Catriona Jamieson. RNA editing enzyme ADAR1 accelerates normal hematopoiesis cell cycle by regulation microRNA biogenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 299. doi:10.1158/1538-7445.AM2017-299