A Split CRISPR-Cpf1 Platform for Inducible Gene Activation.

The CRISPR-Cpf1 also known as Cas12a is an RNA-guided endonuclease similar to CRISPR-Cas9. Combining the CRISPR-Cpf1 with optogenetics technology, we have engineered photoactivatable Cpf1 (paCpf1) to precisely control the genome sequence in a spatiotemporal manner. We also identified spontaneously activated split Cpf1 and thereby developed a potent dCpf1 split activator, which has the potential to activate endogenous target genes. Here we describe a method for optogenetic endogenous genome editing using paCpf1 in mammalian cells. Furthermore, we show a method for endogenous gene activation using dCpf1 split activator in mammalian cells and mice.

A red light-responsive photoswitch for deep tissue optogenetics.

Red light penetrates deep into mammalian tissues and has low phototoxicity, but few optogenetic tools that use red light have been developed. Here we present MagRed, a red light-activatable photoswitch that consists of a red light-absorbing bacterial phytochrome incorporating a mammalian endogenous chromophore, biliverdin and a photo-state-specific binder that we developed using Affibody library selection. Red light illumination triggers the binding of the two components of MagRed and the assembly of split-proteins fused to them. Using MagRed, we developed a red light-activatable Cre recombinase, which enables light-activatable DNA recombination deep in mammalian tissues. We also created red light-inducible transcriptional regulators based on CRISPR-Cas9 that enable an up to 378-fold activation (average, 135-fold induction) of multiple endogenous target genes. MagRed will facilitate optogenetic applications deep in mammalian organisms in a variety of biological research areas.

Optical Control of Genome Editing by Photoactivatable Cas9.

The CRISPR-Cas9 system offers targeted genome manipulation with simplicity. Combining the CRISPR-Cas9 with optogenetics technology, we have engineered photoactivatable Cas9 to precisely control the genome sequence in a spatiotemporal manner. Here we provide a detailed protocol for optogenetic genome editing experiments using photoactivatable Cas9, including that for the generation of guide RNA vectors, light-mediated Cas9 activation, and quantification of genome editing efficiency in mammalian cells.

A slipped-CAG DNA-binding small molecule induces trinucleotide-repeat contractions in vivo.

In many repeat diseases, such as Huntington's disease (HD), ongoing repeat expansions in affected tissues contribute to disease onset, progression and severity. Inducing contractions of expanded repeats by exogenous agents is not yet possible. Traditional approaches would target proteins driving repeat mutations. Here we report a compound, naphthyridine-azaquinolone (NA), that specifically binds slipped-CAG DNA intermediates of expansion mutations, a previously unsuspected target. NA efficiently induces repeat contractions in HD patient cells as well as en masse contractions in medium spiny neurons of HD mouse striatum. Contractions are specific for the expanded allele, independently of DNA replication, require transcription across the coding CTG strand and arise by blocking repair of CAG slip-outs. NA-induced contractions depend on active expansions driven by MutSß. NA injections in HD mouse striatum reduce mutant HTT protein aggregates, a biomarker of HD pathogenesis and severity. Repeat-structure-specific DNA ligands are a novel avenue to contract expanded repeats.

Fine-Tuning of Hydrophobicity in Amphiphilic Polyaspartamide Derivatives for Rapid and Transient Expression of Messenger RNA Directed Toward Genome Engineering in Brain.

Rapid and transient expression of in vitro transcribed mRNA (IVT mRNA) in target cells is a current major challenge in genome engineering therapy. To improve mRNA delivery efficiency, a series of amphiphilic polyaspartamide derivatives were synthesized to contain various hydrophobic moieties with cationic diethylenetriamine (DET) moieties in the side chain and systematically compared as mRNA delivery vehicles (or mRNA-loaded polyplexes). The obtained results demonstrated that the side chain structures of polyaspartamide derivatives were critical for the mRNA delivery efficiency of polyplexes. Interestingly, when the mRNA delivery efficiencies (or the luciferase expression levels in cultured cells) were plotted against an octanol-water partition coefficient (log P) as an indicator of hydrophobicity, a log P threshold was clearly observed to obtain high levels of mRNA expression. Indeed, 3.5 orders of magnitude difference in the expression level is observed between -2.45 and -2.31 in log P. This threshold of log P for the mRNA transfection efficiency apparently correlated with those for the polyplex stability and cellular uptake efficiency. Among the polyaspartamide derivatives with log P > -2.31, a polyaspartamide derivative with 11 residues of 2-cyclohexylethyl (CHE) moieties and 15 residues of DET moieties in the side chains elicited the highest mRNA expression in cultured cells. The optimized polyplex further accomplished highly efficient, rapid, and transient IVT mRNA expression in mouse brain after intracerebroventricular and intrathecal injection. Ultimately, the polyplex allowed for the highly efficient target gene deletion via the expression of Streptococcus pyogenes Cas9 nuclease-coding IVT mRNA in the ependymal layer of ventricles in a reporter mouse model. These results demonstrate the utility of log P driven polymer design for in vivo IVT mRNA delivery.

A split CRISPR-Cpf1 platform for inducible genome editing and gene activation.

The CRISPR-Cpf1 endonuclease has recently been demonstrated as a powerful tool to manipulate targeted gene sequences. Here, we performed an extensive screening of split Cpf1 fragments and identified a pair that, combined with inducible dimerization domains, enables chemical- and light-inducible genome editing in human cells. We also identified another split Cpf1 pair that is spontaneously activated. The newly generated amino and carboxyl termini of the spontaneously activated split Cpf1 can be repurposed as de novo fusion sites of artificial effector domains. Based on this finding, we generated an improved split dCpf1 activator, which has the potential to activate endogenous genes more efficiently than a previously established dCas9 activator. Finally, we showed that the split dCpf1 activator can efficiently activate target genes in mice. These results demonstrate that the present split Cpf1 provides an efficient and sophisticated genome manipulation in the fields of basic research and biotechnological applications.

Inhibition of pre-miRNA-136 processing by Dicer with small molecule BzDANP suggested the formation of ternary complex of pre-miR-136-BzDANP-Dicer.

Small-molecule modulators, along with antisense oligonucleotide, would be powerful tools and potential drug candidates for modulating miRNA-related gene expressions. The mechanism of the inhibitory effect of the C-bulge binding small molecule BzDANP for the Dicer processing reaction of pre-miR-136 was discussed on the data obtained by SPR, NMR, and kinetic analysis for Dicer processing. SPR and NMR analysis showed the preference of BzDANP binding to the C-bulge. Michaelis-Menten analysis suggested the formation of a ternary complex pre-miR-136-BzDANP-Dicer during the Dicer-cleavage reaction of pre-miR-136 in the presence of BzDANP. The inhibitory effect of BzDANP is likely attributed to the slower reaction from the ternary complex than that from the binary pre-miR-136-Dicer complex.

CRISPR-Cas9-based photoactivatable transcription systems to induce neuronal differentiation.

Our improved CRISPR-Cas9-based photoactivatable transcription systems, CPTS2.0 and Split-CPTS2.0, enable high blue-light-inducible activation of endogenous target genes in various human cell lines. We achieved reversible activation of target genes with CPTS2.0 and induced neuronal differentiation in induced pluripotent stem cells (iPSCs) by upregulating NEUROD1 with Split-CPTS2.0.

BzDANP, a Small-Molecule Modulator of Pre-miR-29a Maturation by Dicer.

We here report the synthesis of novel molecule BzDANP having a three-ring benzo[c][1,8]naphthyridine system, the evaluation of its binding properties to a single nucleotide bulge in RNA duplexes, and BzDANP-induced suppression of pre-miR-29a processing by Dicer. BzDANP showed much increased affinity to the bulged RNAs as compared with the parent molecule DANP, which possesses the same hydrogen-bonding surface as BzDANP but in a two-ring [1,8]naphthyridine system. Melting temperature analysis of bulged RNAs showed that BzDANP most effectively stabilized the C-bulged RNA. Dicer-mediated processing of pre-miR-29a was suppressed by BzDANP in a concentration dependent manner. The presence of the C-bulge at the Dicer cleavage site was effective for the suppression of pre-miR-29a processing by BzDANP. These results demonstrated that the small molecule binding to the bulged site in the vicinity of the Dicer cleavage site could be a potential modulator for the maturation of pre-miRNA.

2-Aminophenanthroline dimer stabilized the C-C mismatched duplex DNA.

New ligands with three-ring system for the recognition of a cytosine bulge and a cytosine-cytosine mismatch were designed and synthesized. The 2-amino-1,10-phenanthroline was selected as a recognition unit among the possible three-ring systems of a parent recognition unit of 2-amino-1,8-naphthyridine. The 3-aminopropanamide of 2-amino-1,10-phenanthroline (APM) bound to the cytosine bulge DNA. Other single nucleotide bulges were stabilized by the ligand with much lower efficiency. The dimer APD consisting of two molecules of APM was found to stabilize the C-C mismatch DNA selectively. Structure-activity relationship studies revealed that the APD-binding to the C-C mismatch DNA required both phenanthroline heterocycles in a molecule.

Formation of a ligand-assisted complex of two RNA hairpin loops.

The hairpin structure is one of the most common secondary structures in RNA and holds a central position in the stream of RNA folding from a non-structured RNA to structurally complex and functional ribonucleoproteins. Since the RNA secondary structure is strongly correlated to the function and can be modulated by the binding of small molecules, we have investigated the modulation of RNA folding by a ligand-assisted formation of loop-loop complexes of two RNA hairpin loops. With a ligand (NCT6), designed based on the ligand binding to the G-G mismatches in double-stranded DNA, we successfully demonstrated the formation of both inter- and intra-molecular NCT6-assisted complex of two RNA hairpin loops. NCT6 selectively bound to the two hairpin loops containing (CGG)3 in the loop region. Native polyacrylamide gel electrophoresis analysis of two doubly-labeled RNA hairpin loops clearly showed the formation of intermolecular NCT6-assisted loop-loop complex. Förster resonance energy-transfer studies of RNA constructs containing two hairpin loops, in which each hairpin was labeled with Alexa488 and Cy3 fluorophores, showed the conformational change of the RNA constructs upon binding of NCT6. These experimental data showed that NCT6 simultaneously bound to two hairpin RNAs at the loop region, and can induce the conformational change of the RNA molecule. These data strongly support that NCT6 functions as molecular glue for two hairpin RNAs.

Ligand-inducible formation of RNA pseudoknot.

Here, we demonstrate that a series of naphthyridine derivatives, naphthyridine carbamate tetramer (NCTn), can bind to the RNA CGG/CGG triad comprised of two single-stranded regions of a hairpin loop and a tail. Complete suppression of the binding by a single mutation indicated simultaneous binding of NCTn between hairpin loop and single stranded tail, leading to the formation of NCTn-induced pseudoknot.