A Split CRISPR/Cas13b System for Conditional RNA Regulation and Editing.

The CRISPR/Cas13b system has been demonstrated as a robust tool for versatile RNA studies and relevant applications. New strategies enabling precise control of Cas13b/dCas13b activities and minimal interference with native RNA activities will further facilitate the understanding and regulation of RNA functions. Here, we engineered a split Cas13b system that can be conditionally activated and deactivated under the induction of abscisic acid (ABA), which achieved the downregulation of endogenous RNAs in dosage- and time-dependent manners. Furthermore, an ABA inducible split dCas13b system was generated to achieve temporally controlled deposition of m6A at specific sites on cellular RNAs through conditional assembly and disassembly of split dCas13b fusion proteins. We also showed that the activities of split Cas13b/dCas13b systems can be modulated by light via using a photoactivatable ABA derivative. Overall, these split Cas13b/dCas13b platforms expand the existing repertoire of the CRISPR and RNA regulation toolkit to achieve targeted manipulation of RNAs in native cellular environments with minimal functional disruption to these endogenous RNAs.

Site-Specific m6 A Erasing via Conditionally Stabilized CRISPR-Cas13b Editor.

N6-methyladenosine (m6 A) on RNAs plays an important role in regulating various biological processes and CRIPSR technology has been employed for programmable m6 A editing. However, the bulky size of CRISPR protein and constitutively expressed CRISPR/RNA editing enzymes can interfere with the native function of target RNAs and cells. Herein, we reported a conditional m6 A editing platform (FKBP*-dCas13b-ALK) based on a ligand stabilized dCas13 editor. The inducible expression of this m6 A editing system was achieved by adding or removing the Shield-1 molecule. We further demonstrated that the targeted recruitment of dCas13b-m6 A eraser fusion protein and site-specific m6 A erasing were achieved under the control of Shield-1. Moreover, the release and degradation of dCas13b fusion protein occurred faster than the restoration of m6 A on the target RNAs after Shield-1 removal, which provides an ideal opportunity to study the m6 A function with minimal steric interference from bulky dCas13b fusion protein.

Bifunctional small molecule-oligonucleotide hybrid as microRNA inhibitor.

miRNAs are key regulators of various biological processes. Dysregulation of miRNA is linked to many diseases. Development of miRNA inhibitor has implication in disease therapy and study of miRNA function. The biogenesis pathway of miRNA involves the processing of pre-miRNA into mature miRNA by Dicer enzyme. We previously reported a proximity enabled approach that employs bifunctional small molecules to regulate miRNA maturation through inhibiting the enzymatic activity of Dicer. By conjugating to an RNA targeting unit, an RNase inhibitor could be delivered to the cleavage site of specific pre-miRNA to deactivate the complexed Dicer enzyme. Herein, we expanded this bifunctional strategy by showing that antisense oligonucleotides (ASOs), including morpholinos and γPNAs, could be readily used as the RNA recognition unit to generate bifunctional small molecule-oligonucleotide hybrids as miRNA inhibitors. A systematic comparison revealed that the potency of these hybrids is mainly determined by the RNA binding of the targeting ASO molecules. Since the lengths of the ASO molecules used in this approach were much shorter than commonly used anti-miRNA ASOs, this may provide benefits to the specificity and cellular delivery of these hybrids. We expect that this approach could be complementary to traditional ASO and small molecule based miRNA inhibition and contribute to the study of miRNA.

miRNA inhibition by proximity-enabled Dicer inactivation.

microRNAs (miRNAs) are considered as master regulators of biological processes. Dysregulation of miRNA expression has been implicated in many human diseases. Driven by the key biological roles and the therapeutic potential, developing methods for miRNA regulation has become an intense research area. Due to favorable pharmacological properties, small molecule-based miRNA inhibition emerges as a promising strategy and significant progresses have been made. However, it remains challenging to regulate miRNA using small molecules because of the inherent difficulty in RNA targeting and inhibition. Herein we outline the workflow of generating bifunctional small molecule inhibitors blocking miRNA biogenesis through proximity-enabled inactivation of Dicer, an enzyme required for the processing of precursor miRNA (pre-miRNA) into mature miRNA. By conjugating a weak Dicer inhibitor with a pre-miRNA binder, the inhibitor can be delivered to the Dicer processing site associated with the targeted pre-miRNA, and as a result inhibiting Dicer-mediated pre-miRNA processing. This protocol can be applicable in producing bifunctional inhibitors for different miRNAs.

Chemical and Light Inducible Epigenome Editing.

The epigenome defines the unique gene expression patterns and resulting cellular behaviors in different cell types. Epigenome dysregulation has been directly linked to various human diseases. Epigenome editing enabling genome locus-specific targeting of epigenome modifiers to directly alter specific local epigenome modifications offers a revolutionary tool for mechanistic studies in epigenome regulation as well as the development of novel epigenome therapies. Inducible and reversible epigenome editing provides unique temporal control critical for understanding the dynamics and kinetics of epigenome regulation. This review summarizes the progress in the development of spatiotemporal-specific tools using small molecules or light as inducers to achieve the conditional control of epigenome editing and their applications in epigenetic research.

Cyclic Peptidomimetics as Inhibitor for miR-155 Biogenesis.

miR-155 plays key promoting roles in several cancers and emerges as an important anticancer therapeutic target. However, the discovery of small molecules that target RNAs is challenging. Peptidomimetics have been shown to be a rich source for discovering novel ligands to regulate cellular proteins. However, the potential of using peptidomimetics for RNA targeting is relatively unexplored. To this end, we designed and synthesized members of a novel 320 000 compound macrocyclic peptidomimetic library. An affinity-based screening protocol led to the identification of a pre-miR-155 binder that inhibits oncogenic miR-155 maturation in vitro and in cell and induces cancer cell apoptosis. The results of this investigation demonstrate that macrocyclic peptidomimetics could serve as a new scaffold for RNA targeting.

Self-Reporting Chemically Induced Protein Proximity System Based on a Malachite Green Derivative and the L5** Fluorogen Activating Protein.

A unique chemically induced proximity method is engineered based on mutant antibody VL domain using a fluorogenic malachite green derivative as the inducer, which gives fluorescent signals upon VL domain dimerization while simultaneously inducing downstream biological effects.

Design, synthesis and activity of light deactivatable microRNA inhibitor.

miRNAs are key cellular regulators and their dysregulation is associated with many human diseases. They are usually produced locally in a spatiotemporally controlled manner to target mRNAs and regulate gene expression. Thus, developing chemical tools for manipulating miRNA with spatiotemporal precise is critical for studying miRNA. Herein, we designed a strategy to control miRNA biogenesis with light controllable inhibitor targeting the pre-miRNA processing by Dicer. By conjugating two non-inhibiting units, a low affinity Dicer inhibitor and a pre-miRNA binder, through a photocleavable linker, the bifunctional molecule obtained could inhibit miRNA production. Taking advantage of the photocleavable property of the linker, the bifunctional inhibitor can be fragmented into separate non-inhibiting units and therefore be deactivated by light. We expect that this strategy could be applied to generate chemical biological tools that allow light-mediated spatiotemporal control of miRNA maturation and contribute to the study of miRNA function.

Regulating miRNA-21 Biogenesis By Bifunctional Small Molecules.

We report a new strategy to regulate microRNAs (miRNAs) biogenesis by using bifunctional small molecules that consist of a pre-miRNA binding unit connected by a linker to a Dicer inhibiting unit. In this effort, fluorescence polarization-based screening was used to identify neomycin as a pre-miR-21 binding ligand. Although neomycin cannot inhibit miR-21 maturation, linking it to the RNase inhibitor 1 forms the bifunctional conjugate 7A, which inhibits the production of miR-21. We expect that this strategy will be applicable to design other molecules for miRNA regulation.

Chemically Controlled Epigenome Editing through an Inducible dCas9 System.

Although histone modifications are associated with gene activities, studies of their causal relationships have been difficult. For this purpose, we developed an inducible system integrating dCas9-based targeting and chemically induced proximity technologies to allow small molecule induced recruitment of P300 acetyltransferase and the acetylation of H3K27 at precise gene loci in cells. Employing the new technique, we elucidated the temporal order of histone acetylation and gene activation, as well as the stability of the installed histone modification.

Reaction-Based "Off-On" Fluorescent Probe Enabling Detection of Endogenous Labile Fe(2+) and Imaging of Zn(2+)-induced Fe(2+) Flux in Living Cells and Elevated Fe(2+) in Ischemic Stroke.

Fluorogenic sensors capable of spatiotemporally detecting Fe(2+) in biological systems are highly valuable in the study of iron biology. Toward this end, a new "off-on" Fe(2+)-selective fluorescent probe has been developed by incorporating an Fe(2+)-induced N-O cleavage of acylated hydroxylamine moiety into the naphthalimide fluorophore. The probe displays facile response (within 15 min) and good selectivity toward Fe(2+) with >27-fold enhancement of fluorescence intensity and high sensitivity of as low as 0.5 µM with a noticeable 3-fold fluorescence enhancement. These features of the probe have been transformed into in the convenient detection of endogenous, basal level of labile Fe(2+) pools in living cells. Furthermore, we have demonstrated the capacity of the probe for the studies of important Fe(2+) related biological functions. It can respond to the Zn(2+)-induced Fe(2+) flux, an important event observed in stroke, and facilely detect the elevated level of Fe(2+) in the brain tissue of a rat undergoing ischemic stroke at the ischemic site.

Light control of cellular processes by using photocaged abscisic acid.

Abscisic acid (ABA) was chemically modified with a photocaging group to promote photo-induced protein dimerization. This photocontrolled chemically induced dimerization (CID) method based on caged ABA enables dose-dependent light regulation of cellular processes, including transcription, protein translocation, signal transduction, and cytoskeletal remodeling, without the need to perform extensive protein engineering. Caged ABA can be easily modified to respond to different wavelengths of light. Consequently, this strategy should be applicable to the design of light-regulated protein dimerization systems and potentially be used orthogonally with other light-controlled CID systems.

Induced proximity labeling and editing for epigenetic research.

Epigenetic regulation plays a pivotal role in various biological and disease processes. Two key lines of investigation have been pursued that aim to unravel endogenous epigenetic events at particular genes (probing) and artificially manipulate the epigenetic landscape (editing). The concept of induced proximity has inspired the development of powerful tools for epigenetic research. Induced proximity strategies involve bringing molecular effectors into spatial proximity with specific genomic regions to achieve the probing or manipulation of local epigenetic environments with increased proximity. In this review, we detail the development of induced proximity methods and applications in shedding light on the intricacies of epigenetic regulation.

A CRISPR-based Strategy for Temporally Controlled Site-Specific Editing of RNA Modifications.

Chemical modifications on RNA play important roles in regulating its fate and various biological activities. However, the impact of RNA modifications varies depending on their locations on different transcripts and cells/tissues contexts; available tools to dissect context-specific RNA modifications are still limited. Herein, we report the detailed protocol for using a chemically inducible and reversible platform to achieve site-specific editing of the chosen RNA modification in a temporally controlled manner by integrating the clustered regularly interspaced short palindromic repeats (CRISPR) technology and the abscisic acid (ABA)-based chemically induced proximity (CIP) system. The procedures were demonstrated using the example of inducible and reversible N6-methyladenosine (m6A) editing and the evaluation of its impact on RNA properties with ABA addition and reversal with the control of ABA or light.

A theranostic abscisic acid-based molecular glue.

Molecular glues, capable of selectively controlling the interactions between specific pairs or groups of proteins and the associated downstream effects, have become a promising strategy for manipulating cellular functions and developing novel therapies for human diseases. Theranostics with both diagnostic and therapeutic capabilities acting at disease sites has become a powerful tool to achieve both functions simultaneously with high precision. To selectively activate molecular glues at the desired site and monitor the activation signals at the same time, here we report an unprecedented theranostic modular molecular glue platform integrating signal sensing/reporting and chemically induced proximity (CIP) strategies. We have demonstrated for the first time the integration of imaging and activation capacity with a molecular glue on the same platform to create a theranostic molecular glue. A theranostic molecular glue ABA-Fe(ii)-F1 was rationally designed by conjugating a NIR fluorophore dicyanomethylene-4H-pyran (DCM) with a CIP inducer abscisic acid (ABA) using a unique carbamoyl oxime linker. We have also engineered a new version of ABA-CIP with an enhanced ligand-responding sensitivity. We have validated that the theranostic molecular glue can sense Fe2+ and produce turn-on NIR fluorescence for monitoring as well as releasing the active inducer ligand to control cellular functions including gene expression and protein translocation. This novel molecular glue strategy paves the way to building a new class of molecular glues with theranostic capacity for research and biomedical applications.

Inducible and reversible RNA N6-methyladenosine editing.

RNA modifications, including N6-methyladenosine (m6A), have been reported to regulate fundamental RNA processes and properties, and directly linked to various human diseases. Methods enabling temporal and transcript/locus-specific editing of specific RNA modifications are essential, but still limited, to dissect the dynamic and context-dependent functions of these epigenetic modifications. Here, we develop a chemically inducible and reversible RNA m6A modification editing platform integrating chemically induced proximity (CIP) and CRISPR methods. We show that m6A editing can be temporally controlled at specific sites of individual RNA transcripts by the addition or removal of the CIP inducer, abscisic acid (ABA), in the system. By incorporating a photo-caged ABA, a light-controlled version of m6A editing platform can be developed. We expect that this platform and strategy can be generally applied to edit other RNA modifications in addition to m6A.

Investigating crosstalk between H3K27 acetylation and H3K4 trimethylation in CRISPR/dCas-based epigenome editing and gene activation.

Epigenome editing methods enable the precise manipulation of epigenetic modifications, such as histone posttranscriptional modifications (PTMs), for uncovering their biological functions. While histone PTMs have been correlated with certain gene expression status, the causalities remain elusive. Histone H3 Lysine 27 acetylation (H3K27ac) and histone H3 Lysine 4 trimethylation (H3K4me3) are both associated with active genes, and located at active promoters and enhancers or around transcriptional start sites (TSSs). Although crosstalk between histone lysine acetylation and H3K4me3 has been reported, relationships between specific epigenetic marks during transcriptional activation remain largely unclear. Here, using clustered regularly interspaced short palindromic repeats (CRISPR)/dCas-based epigenome editing methods, we discovered that the ectopic introduction of H3K27ac in the promoter region lead to H3K4me3 enrichment around TSS and transcriptional activation, while H3K4me3 installation at the promoter cannot induce H3K27ac increase and failed to activate gene expression. Blocking the reading of H3K27ac by BRD proteins using inhibitor JQ1 abolished H3K27ac-induced H3K4me3 installation and downstream gene activation. Furthermore, we uncovered that BRD2, not BRD4, mediated H3K4me3 installation and gene activation upon H3K27ac writing. Our studies revealed the relationships between H3K27ac and H3K4me3 in gene activation process and demonstrated the application of CRISPR/dCas-based epigenome editing methods in elucidating the crosstalk between epigenetic mechanisms.

Chemical Inducible dCas9-Guided Editing of H3K27 Acetylation in Mammalian Cells.

The ability to edit specific epigenetic modifications at defined gene loci is pivotal to understand the biological function of these epigenetic marks. Here we describe a new inducible method to integrate the dCas9-based genome targeting with abscisic acid (ABA)-based chemically induced proximity (CIP) technologies to modify histone tail modifications at specific genome loci in living cells. ABA leads to rapid hetero-dimerization of the PYL and ABI proteins, which can be individually fused to dCas9 and a histone-modifying enzyme core domain. In the presence of ABA and locus-specific sgRNAs, this histone-modifying activity can be recruited to a specific genome locus to achieve histone editing with perfect temporal control. Herein, we describe the use of this technique in HEK293T cells to control the recruitment of the p300 acetyltransferase core domain to the human IL1RN locus to ectopically increase the acetylation of H3K27 and induce the expression of IL1RN gene.

A chemically induced proximity system engineered from the plant auxin signaling pathway.

Methods based on chemically induced proximity (CIP) serve as powerful tools to control cellular processes in a temporally specific manner. To expand the repertoire of CIP systems available for studies of cellular processes, we engineered the plant auxin signaling pathway to create a new indole-3-acetic acid (IAA) based CIP method. Auxin-induced protein degradation that occurs in the natural pathway was eliminated in the system. The new IAA based method is both readily inducible and reversible, and used to control the production of therapeutic proteins that induced the apoptosis of cancer cells. The approach is also orthogonal to existing CIP systems and used to construct a biological Boolean logic gate controlling gene expression system. We believe that the new CIP method will be applicable to the artificial control and dissection of complex cellular functions.