Ribozyme-Mediated Knockdown of lncRNA Gene Expression in Drosophila

The current toolkit for genetic manipulation in the model animal Drosophila melanogaster is extensive and versatile but not without its limitations. Here, we report a powerful and heritable method to knockdown gene expression in D. melanogaster using the self-cleaving N79 hammerhead ribozyme, a modification of a naturally occurring ribozyme found in the parasite Schistosoma mansoni. A 111-bp ribozyme cassette, consisting of the N79 ribozyme surrounded by insulating spacer sequences, was inserted into 4 independent long noncoding RNA genes as well as the male-specific splice variant of doublesex using scarless CRISPR/Cas9-mediated genome editing. Ribozyme-induced RNA cleavage resulted in robust destruction of 3' fragments typically exceeding 90%. Single molecule RNA fluorescence in situ hybridization results suggest that cleavage and destruction can even occur for nascent transcribing RNAs. Knockdown was highly specific to the targeted RNA, with no adverse effects observed in neighboring genes or the other splice variants. To control for potential effects produced by the simple insertion of 111 nucleotides into genes, we tested multiple catalytically inactive ribozyme variants and found that a variant with scrambled N79 sequence best recapitulated natural RNA levels. Thus, self-cleaving ribozymes offer a novel approach for powerful gene knockdown in Drosophila, with potential applications for the study of noncoding RNAs, nuclear-localized RNAs, and specific splice variants of protein-coding genes.

Thousands of long non-coding RNAs (lncRNAs) have been identified in mammalian cells. Some have important functions and their dysregulation can contribute to a variety of disease states. However, most lncRNAs have not been functionally characterized. Complicating their study, lncRNAs have widely varying subcellular distributions: some reside predominantly in the nucleus, the cytoplasm or in both compartments. One method to query function is to suppress expression and examine the resulting phenotype. Methods to suppress expression of mRNAs include antisense oligonucleotides (ASOs) and RNA interference (RNAi). Antisense and RNAi-based gene-knockdown methods vary in efficacy between different cellular compartments. It is not known if this affects their ability to suppress lncRNAs. To address whether localization of the lncRNA influences susceptibility to degradation by either ASOs or RNAi, nuclear lncRNAs (MALAT1 and NEAT1), cytoplasmic lncRNAs (DANCR and OIP5-AS1) and dual-localized lncRNAs (TUG1, CasC7 and HOTAIR) were compared for knockdown efficiency. We found that nuclear lncRNAs were more effectively suppressed using ASOs, cytoplasmic lncRNAs were more effectively suppressed using RNAi and dual-localized lncRNAs were suppressed using both methods. A mixed-modality approach combining ASOs and RNAi reagents improved knockdown efficacy, particularly for those lncRNAs that localize to both nuclear and cytoplasmic compartments.

Investigations of CRISPR gene knockout editing profiles have contributed to enhanced precision of editing outcomes. However, for homology-directed repair (HDR) in particular, the editing dynamics and patterns in clinically relevant cells, such as human iPSCs and primary T cells, are poorly understood. Here, we explore the editing dynamics and DNA repair profiles after the delivery of Cas9-guide RNA ribonucleoprotein (RNP) with or without the adeno-associated virus serotype 6 (AAV6) as HDR donors in four cell types. We show that editing profiles have distinct differences among cell lines. We also reveal the kinetics of HDR mediated by the AAV6 donor template. Quantification of T50 (time to reach half of the maximum editing frequency) indicates that short indels (especially +A/T) occur faster than longer (>2 bp) deletions, while the kinetics of HDR falls between NHEJ (non-homologous end-joining) and MMEJ (microhomology-mediated end-joining). As such, AAV6-mediated HDR effectively outcompetes the longer MMEJ-mediated deletions but not NHEJ-mediated indels. Notably, a combination of small molecular compounds M3814 and Trichostatin A (TSA), which potently inhibits predominant NHEJ repairs, leads to a 3-fold increase in HDR efficiency.

Systemic uncontrolled inflammatory response, also termed as sepsis, is responsible for many mortalities. Bacterial endotoxin, lipopolysaccharide (LPS), is a major cause of sepsis in endothelial cells. Even though a lot of research has been done to define underlying mechanisms of LPS induced sepsis, the role of long non-coding RNAs (lncRNAs), a group of >200 kb RNAs in sepsis is not well-defined. Expression of pro-inflammatory mediators IL6, ICAM1, and VCAM1 (which encodes interleukin-6, intercellular adhesion molecule-1, and vascular cell adhesion molecule-1, respectively) were determined following LPS treatment of human dermal microvascular endothelial cells (HMECs) for 24 h to confirm sepsis induction. RNA immunoprecipitation (RIP) analysis was performed using the chromatin modifying proteins (CMPs), heterogeneous nuclear ribonucleoprotein (hnRNP) K and corepressors of the RE-1 silencing transcription factor (coREST) as individual baits. Quantitative real time polymerase chain reaction (qRT-PCR) was performed on RNA isolated from immunoprecipitated pellets for six different lncRNAs. The effect of the differentially expressed lncRNAs were determined by ectopic overexpression of the lncRNAs before induction of sepsis. Expression of IL6, ICAM1, and VCAM1 were significantly upregulated following treatment of the HMECs with LPS for 24 h confirming induction of sepsis. RIP and qRT-PCR analysis revealed that the lncRNAs HULC, UCA1, and MALAT-1 were significantly enriched with the CMPs after sepsis. RNA interference using siRNAs targeting HULC and UCA1, but not MALAT-1, decreased the expression of IL6, ICAM1, and VCAM1 to endogenous levels. Our results were further validated in an in vivo model of sub-lethal LPS-induced sepsis, whereby siRNA mediated knockdown of UCA1 and HULC lncRNAs prevented induction of VCAM1, ICAM1, and IL6, as assayed by immunohistochemistry. Cumulatively, these results suggest that LPS induced in vitro sepsis in endothelial cells and induction of pre-inflammatory mediators are at least in part due to increased expression of the UCA1 and HULC lncRNAs.

Exercise-Induced Long Noncoding RNAs As New Players in Cardiac Hypertrophy.

To study the expression activity of various vectors containing anti-caspase-3 ribozyme cassettes in vivo, and to further study the role of caspas-3 in the apoptotic pathway, we constructed anti-caspase-3 hammerhead ribozyme embedded into the human snRNA U6, and detected the activity of the ribozyme in vitro and in vivo. Meanwhile we compared it with the self-cleaving hammerhead ribozymes that we previously studied, and with the general ribozyme, cloned into RNA polymerase II expression systems. The results showed that the three ribozymes, p1.5RZ107, pRZ107 and pU6RZ107 had the correct structure, and that they could cleave caspase-3 mRNA exactly to produce two fragments: 143nt/553nt. p1.5RZ107 has the highest cleavage efficiency in vitro, almost 80%. However, the U6 chimeric ribozyme, pU6RZ107, has the highest cleavage activity in vivo, almost to 65%, though it has lower cleavage activity in vitro. The cleavage results demonstrated that the pU6RZ107, the U6 chimeric ribozyme, could more efficiently express and downregulate the level of caspase-3 in vivo, and the ribozyme could provide an alternative approach to the research into the mechanism of apoptosis and human gene therapy also.

RNA interference (RNAi) is an important tool for studying gene function and genetic networks. Double-stranded RNA (dsRNA) triggers RNAi that selectively silences gene expression mainly by degrading target mRNA sequences. Short interfering RNA, short hairpin RNA (shRNA), long dsRNA, and microRNA-based shRNA (shRNAmir) are four different types of dsRNA that have been widely used to silence gene expression in cultured cells, tissues, organs, and organisms. Long dsRNAs are usually 200–500 nucleotides in length and can selectively suppress expression of target genes in Caenorhabditis elegans and Drosophila but not in mammals due to unwanted non-specific knockdown. Thus, multiple attempts have been made to synthesize, express, and deliver short dsRNAs that specifically silence target genes in mammals. We describe a method for constructing an RNAi library by converting cDNAs into shRNAmir30 sequences by sequential treatment with different enzymes and affinity purification of biotin- or digoxygenin-labeled DNA fragments. We also developed a system to generate stable cell lines that uniformly express shRNAmir30s and fluorescence reporters by Cre recombinase-dependent site-specific recombination. Thus, combined with the RNAi library, this system facilitates screening for potent RNAi sequences that strongly suppress expression of target genes.

siRNA-optimized Modifications for Enhanced In Vivo Activity

Short hairpin RNAs (shRNAs) can induce gene silencing via the RNA interference (RNAi) mechanism. We designed an alternative shRNA molecule with a relatively short base-paired stem that bypasses Dicer and instead is processed by the Argonaute 2 (Ago2) protein into a single guide RNA strand that effectively induces RNAi. We called these molecules AgoshRNAs. Active anti-HIV AgoshRNAs were developed, but their RNAi activity was generally reduced compared with the matching shRNAs. In an attempt to further optimize the AgoshRNA design, we inserted several self-cleaving ribozymes at the 3′ terminus of the transcribed AgoshRNA and evaluated the impact on AgoshRNA processing and activity. The hepatitis delta virus (HDV) ribozyme is efficiently removed from the transcribed AgoshRNAs and generates a uniform 3′ overhang, which translates into the enhanced antiviral activity of these molecules.

The type2 CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR- associated protein 9) is an efficient RNA-guided genome-editing technique. Guided by sgRNA, the Cas9 endonuclease generates site-specific double-stranded breaks (DSB) at specific site, which is amenable to repair by homology-directed repair (HDR) to generate a designed knock-out or knock-in transgene. In combination with CRISPR/Cas9 and Cre/loxP or FLP/FRT system, efficient gene targeting can be achieved, and meanwhile screening markers introduced can be readily removed except a 34-base pair residual fragment. Thus, difficulties remain in accurate editing of the genome without introducing any extraneous sequences. In human induced pluripotent stem cells (iPSCs), a two-step strategy has been developed using CRISPR/Cas9 and the piggyBac system to establish a seamless genomic editing, in which CRISPR/Cas9 is initially used to introduce mutations along with screening markers by HDR, then the markers are precisely excised by piggyBac transposase. Using this strategy, we have successfully transformed the tyrosine to cysteine at position 21 within the 18th exon of the CG4894 gene in the Drosophila genome without introducing any extraneous sequence. Hence, this strategy provides more options for precise and seamless editing of the Drosophila genome.

Self-cleaving ribozymes are biologically relevant RNA molecules which catalyze site-specific cleavage of the phosphodiester backbone. Gathering knowledge of their three-dimensional structures is critical toward an in-depth understanding of their function and chemical mechanism. Equally important is collecting information on the folding process and the inherent dynamics of a ribozyme fold. Over the past years, Selective-2′-Hydroxyl Acylation analyzed by Primer Extension (SHAPE) turned out to be a significant tool to probe secondary and tertiary interactions of diverse RNA species at the single nucleotide level under varying environmental conditions. Small self-cleaving ribozymes, however, have not been investigated by this method so far. Here, we describe SHAPE probing of pre-catalytic folds of the recently discovered ribozyme classes twister, twister-sister (TS), pistol and hatchet. The study has implications on Mg2+-dependent folding and reveals potentially dynamic residues of these ribozymes that are otherwise difficult to identify. For twister, TS and pistol ribozymes the new findings are discussed in the light of their crystal structures, and in case of twister also with respect to a smFRET folding analysis. For the hatchet ribozyme where an atomic resolution structure is not yet available, the SHAPE data challenge the proposed secondary structure model and point at selected residues and putative long-distance interactions that appear crucial for structure formation and cleavage activity.

Once again, the fruit fly has proven an invaluable tool for revealing information about the immune responses involving the transcription factor nuclear factor κB (NF-κB), as Beutler and Moresco emphasize. Goto et al . performed a genome-wide RNA interference (RNAi) screen in cultured Drosophila S2 cells for genes required for NF-κB-dependent gene expression and found the gene CG8580 , which they renamed Akirin (for the Japanese akiraka ni suru, meaning "making things clear"). The authors determined that Drosophila has a single Akirin gene, which is essential for viability, and there are two in mammals ( Akirin1 , which is not essential for viability in mice, and Akirin2 , which is). Transfection experiments with tagged versions of the proteins indicated that Akirins are localized to the nucleus, and the nuclear localization signal is the only recognizable motif in these proteins. S2 cells in which Akirin was knocked down showed defective activation of gene expression in response to stimuli that activate the Imd pathway, which mediates immune response to Gram-negative bacteria through the activation of the NF-κB homolog Relish. In contrast, the response to signaling through the Toll pathway, which mediates the response to Gram-positive bacteria through the activation of the NF-κB homolog Dif was not affected. Epistasis experiments placed Akirin at the level of Relish in the Imd pathway. In flies, knockdown of Akirin led to decreased survival after Gram-negative bacterial infection. Analysis of Akirin1 or Akirin2 knockout mouse embryo fibroblasts (MEFs) suggested that Akirin2 was the homolog involved in NF-κB signaling downstream of Toll-like receptors (TLRs) or the tumor necrosis factor (TNF) receptor. Furthermore, the human Akirin2 protein rescued S2 cells from defective Imd signaling caused by knockdown of endogenous Akirin. MEFs deficient for Akirin2 showed impaired expression of a subset of NF-κB-regulated genes, yet stimulation of NF-κB DNA binding and degradation of the inhibitor protein IκBα was not impaired. These results suggest that in mice, as in the fly, Akirin2 acts at the same level as NF-κB, and it will be interesting to see whether these two proteins form a complex, as has been recently reported for ribosomal protein S3 and NF-κB (see related resource). A. Goto, K. Matsushita, V. Gesellchen, L. El Chamy, D. Kuttenkeuler, O. Takeuchi, J. A. Hoffmann, S. Akira, M. Boutros, J.-M. Reichhart, Akirins are highly conserved nuclear proteins required for NF-κB-dependent gene expression in drosophila and mice. Nat. Immunol. 9 , 97-104 (2008). [PubMed] B. Beutler, E. M. Y. Moresco, Akirins versus infection. Nat. Immunol. 9 , 7-9 (2008). [PubMed]

Background: Gene correction via homology directed repair (HDR) in patient-derived induced pluripotent stem (iPS) cells for regenerative medicine are becoming a more realistic approach to develop personalized and mutation-specific therapeutic strategies due to current developments in gene editing and iPSC technology. Cystic fibrosis (CF) is the most common inherited disease in the Caucasian population, caused by mutations in the CF transmembrane conductance regulator (CFTR) gene. Since CF causes significant multi-organ damage and with over 2,000 reported CFTR mutations, CF patients could be one prominent population benefiting from gene and cell therapies. When considering gene-editing techniques for clinical applications, seamless gene corrections of the responsible mutations, restoring native “wildtype” DNA sequence without remnants of drug selectable markers or unwanted DNA sequence changes, would be the most desirable approach.Result: The studies reported here describe the seamless correction of the W1282X CFTR mutation using CRISPR/Cas9 nickases (Cas9n) in iPS cells derived from a CF patient homozygous for the W1282X Class I CFTR mutation. In addition to the expected HDR vector replacement product, we discovered another class of HDR products resulting from vector insertion events that created partial duplications of the CFTR exon 23 region. These vector insertion events were removed via intrachromosomal homologous recombination (IHR) enhanced by double nicking with CRISPR/Cas9n which resulted in the seamless correction of CFTR exon 23 in CF-iPS cells.Conclusion: We show here the removal of the drug resistance cassette and generation of seamless gene corrected cell lines by two independent processes: by treatment with the PiggyBac (PB) transposase in vector replacements or by IHR between the tandemly duplicated CFTR gene sequences.

Most new technologies for manipulating gene expression in mammalian cells are accepted at a relatively slow pace. Occasionally, however, a new technology is so robust and fills such a critical niche that its adoption is widespread and rapid. Fifteen years ago, duplex RNAs were such a technology. RNA interference (RNAi) in mammalian cells was first demonstrated in 2001 (1) and within 2 y RNAi was a commonly used tool throughout industry and academia. RNAi is making its way into clinical trials as a potential therapeutic as challenges in delivery to relevant tissues begin to be overcome (2⇓–4). More recently, another revolution in biology appears to be emerging, powered by bacterial type II clustered, regularly interspaced, short palindromic repeats (CRISPR)-associated (Cas) systems. In PNAS, Rahdar et al. take a step toward a strategy that combines genetic and synthetic approaches for delivery of active CRISPR-Cas in vivo (5). CRISPR-Cas is based on a natural bacterial defense mechanism for controlling pathogens (6). The realization that CRISPR-Cas can efficiently direct cleavage of double-stranded DNA in diverse biological systems has rapidly transformed it into a deft tool for genome editing (7⇓–9). The most popular CRISPR-Cas system makes use of the Cas9 endonuclease from Streptococcus pyogenes . Cas9 binds a short 42-nt-long CRISPR RNA (crRNA) and an 80-nt transactivating crRNA (tracrRNA). The crRNA has a variable guide sequence that directs Cas9 endonuclease activity to sequence-specifically cut both strands of a DNA target. Cleavage typically introduces insertions or deletions through errors in natural DNA repair mechanisms. The presence of an appropriate donor DNA can also result in accurate insertion of new sequences through homology-directed repair. By this method, permanent changes to the genome are accomplished. Thus, CRISPR-Cas represents … [↵][1]1To whom correspondence may be addressed. Email: ktgagnon{at}siu.edu or david.corey{at}utsouthwestern.edu. [1]: #xref-corresp-1-1

Long noncoding RNAs (lncRNAs) play key regulatory roles in gene expression at the transcriptional level. Experimental evidence has established that a substantial fraction of lncRNA preferentially accumulates in the nucleus. For analysis of the function of nuclear lncRNAs, it is important to achieve efficient knockdown of these transcripts inside the nucleus. In contrast to the use of RNA interference, a technology that depends on the cytoplasmic silencing machinery, an antisense oligonucleotide (ASO) technology can achieve RNA knockdown by recruiting RNase H to the RNA-DNA duplexes for nuclear RNA cleavage. Unlike the use of CRISPR-Cas tools for genome engineering, where possible alterations in the chromatin state can occur, ASOs allow the efficient knockdown of nuclear transcripts without modifying the genome. Nevertheless, one of the major obstacles to ASO-mediated knockdown is its transitory effect. For the study of long-lasting effects of lncRNA silencing, maintaining efficient knockdown for a long time is needed. In this study, a protocol was developed to achieve a knockdown effect for over 21 days. The purpose was to evaluate the cis-regulatory effects of lncRNA knockdown on the adjacent coding gene RFC4, which is related to chromosomal instability, a condition that is observed only through time and cell aging. Two different human cell lines were used: PrEC, normal primary prostate epithelial cells, and HCT116, an epithelial cell line isolated from colorectal carcinoma, achieving successful knockdown in the assayed cell lines.