Pseudoknot-targeting Cas13b combats SARS-CoV-2 infection by suppressing viral replication.

CRISPR-Cas13-mediated viral genome targeting is a novel strategy for defending against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants. Here, we generated mRNA-encoded Cas13b targeting the open reading frame 1b (ORF1b) region to effectively degrade the RNA-dependent RNA polymerase gene. Of the 12 designed CRISPR RNAs (crRNAs), those targeting the pseudoknot site upstream of ORF1b were found to be the most effective in suppressing SARS-CoV-2 propagation. Pseudoknot-targeting Cas13b reduced expression of the spike protein and attenuated viral replication by 99%. It also inhibited the replication of multiple SARS-CoV-2 variants, exhibiting broad potency. We validated the therapeutic efficacy of this system in SARS-CoV-2-infected hACE2 transgenic mice, demonstrating that crRNA treatment significantly reduced viral titers. Our findings suggest that the pseudoknot region is a strategic site for targeted genomic degradation of SARS-CoV-2. Hence, pseudoknot-targeting Cas13b could be a breakthrough therapy for overcoming infections by SARS-CoV-2 or other RNA viruses.

Optogenetic activation of intracellular antibodies for direct modulation of endogenous proteins.

Intracellular antibodies have become powerful tools for imaging, modulating and neutralizing endogenous target proteins. Here, we describe an optogenetically activated intracellular antibody (optobody) consisting of split antibody fragments and blue-light inducible heterodimerization domains. We expanded this optobody platform by generating several optobodies from previously developed intracellular antibodies, and demonstrated that photoactivation of gelsolin and ß2-adrenergic receptor (ß2AR) optobodies suppressed endogenous gelsolin activity and ß2AR signaling, respectively.

Programmable RNA base editing with photoactivatable CRISPR-Cas13.

CRISPR-Cas13 is widely used for programmable RNA interference, imaging, and editing. In this study, we develop a light-inducible Cas13 system called paCas13 by fusing Magnet with fragment pairs. The most effective split site, N351/C350, was identified and found to exhibit a low background and high inducibility. We observed significant light-induced perturbation of endogenous transcripts by paCas13. We further present a light-inducible base-editing system, herein called the padCas13 editor, by fusing ADAR2 to catalytically inactive paCas13 fragments. The padCas13 editor enabled reversible RNA editing under light and was effective in editing A-to-I and C-to-U RNA bases, targeting disease-relevant transcripts, and fine-tuning endogenous transcripts in mammalian cells in vitro. The padCas13 editor was also used to adjust post-translational modifications and demonstrated the ability to activate target transcripts in a mouse model in vivo. We therefore present a light-inducible RNA-modulating technique based on CRISPR-Cas13 that enables target RNAs to be diversely manipulated in vitro and in vivo, including through RNA degradation and base editing. The approach using the paCas13 system can be broadly applicable to manipulating RNA in various disease states and physiological processes, offering potential additional avenues for research and therapeutic development.

Optogenetic Activation of Intracellular Nanobodies.

Intracellular antibody fragments such as nanobodies and scFvs are powerful tools for imaging and for modulating and neutralizing endogenous target proteins. Optogenetically activated intracellular antibodies (optobodies) constitute a light-inducible system to directly control intrabody activities in cells, with greater spatial and temporal resolution than intracellular antibodies alone. Here, we describe optogenetic and microscopic methods to activate optobodies in cells using a confocal microscope and an automated fluorescence microscope. In the protocol, we use the examples of an optobody targeting green fluorescent protein and an optobody that inhibits the endogenous gelsolin protein.

Mutations in FAM50A suggest that Armfield XLID syndrome is a spliceosomopathy.

Intellectual disability (ID) is a heterogeneous clinical entity and includes an excess of males who harbor variants on the X-chromosome (XLID). We report rare FAM50A missense variants in the original Armfield XLID syndrome family localized in Xq28 and four additional unrelated males with overlapping features. Our fam50a knockout (KO) zebrafish model exhibits abnormal neurogenesis and craniofacial patterning, and in vivo complementation assays indicate that the patient-derived variants are hypomorphic. RNA sequencing analysis from fam50a KO zebrafish show dysregulation of the transcriptome, with augmented spliceosome mRNAs and depletion of transcripts involved in neurodevelopment. Zebrafish RNA-seq datasets show a preponderance of 3' alternative splicing events in fam50a KO, suggesting a role in the spliceosome C complex. These data are supported with transcriptomic signatures from cell lines derived from affected individuals and FAM50A protein-protein interaction data. In sum, Armfield XLID syndrome is a spliceosomopathy associated with aberrant mRNA processing during development.

Noninvasive optical activation of Flp recombinase for genetic manipulation in deep mouse brain regions.

Spatiotemporal control of gene expression or labeling is a valuable strategy for identifying functions of genes within complex neural circuits. Here, we develop a highly light-sensitive and efficient photoactivatable Flp recombinase (PA-Flp) that is suitable for genetic manipulation in vivo. The highly light-sensitive property of PA-Flp is ideal for activation in deep mouse brain regions by illumination with a noninvasive light-emitting diode. In addition, PA-Flp can be extended to the Cre-lox system through a viral vector as Flp-dependent Cre expression platform, thereby activating both Flp and Cre. Finally, we demonstrate that PA-Flp-dependent, Cre-mediated Cav3.1 silencing in the medial septum increases object-exploration behavior in mice. Thus, PA-Flp is a noninvasive, highly efficient, and easy-to-use optogenetic module that offers a side-effect-free and expandable genetic manipulation tool for neuroscience research.