Assa, D. | bioRxiv Science

bioRxiv2026

Chemically modified CRISPR enzymes for multi-organ genome editing in vivo

Delivery remains the main obstacle to the development of in vivo genome editing therapies. CRISPR ribonucleoproteins confer high editing activity with transient exposure but lack intrinsic cell entry and targeting. Here we introduce PERCEPT, a delivery platform featuring reversible, covalent modification of CRISPR enzymes. PERCEPT enables modular installation of shielding polymers, amphiphilic delivery peptides and targeting ligands, allowing traceless cytosolic release of the native editor. A formulation incorporating an amphiphilic delivery peptide and the neuron-targeting ligand TET1 edited approximately 56% of striatal volume and 78% of neurons within edited regions after local striatal administration. In the R6/2 Huntington's disease model, PERCEPT mediated targeted editing of the mutant human HTT transgene, reducing mutant huntingtin aggregate burden and shifting local transcriptional programs away from inflammatory and injury-associated states. Tissue-adapted formulations edited 53% of Muller glia following intravitreal delivery, increased skeletal muscle reporter fluorescence tenfold versus unconjugated control following intramuscular injection, and enabled lung airway epithelial cell that persisted for three months following intranasal delivery. Rapid screening revealed that local anatomical and cellular barriers require distinct surface display for optimal editing. These results establish reversible chemical modification as a general strategy for adapting CRISPR enzyme delivery across multiple target tissues in vivo.

bioengineering↗

bioRxiv2023

Chemical stimuli override a temperature-dependent morphological program by reprogramming the transcriptome of a fungal pathogen.

The human fungal pathogen Histoplasma changes its morphology in response to temperature. At 37{degrees}C it grows as a budding yeast whereas at room temperature it transitions to hyphal growth. Prior work has demonstrated that 15-20% of transcripts are temperature-regulated, and that transcription factors Ryp1-4 are necessary to establish yeast growth. However, little is known about transcriptional regulators of the hyphal program. To identify TFs that regulate filamentation, we utilize chemical inducers of hyphal growth. We show that addition of cAMP analogs or an inhibitor of cAMP breakdown overrides yeast morphology, yielding inappropriate hyphal growth at 37{degrees}C. Additionally, butyrate supplementation triggers hyphal growth at 37{degrees}C. Transcriptional profiling of cultures filamenting in response to cAMP or butyrate reveals that a limited set of genes respond to cAMP while butyrate dysregulates a larger set. Comparison of these profiles to previous temperature- or morphology-regulated gene sets identifies a small set of morphology-specific transcripts. This set contains 9 TFs of which we characterized three, STU1, FBC1, and PAC2, whose orthologs regulate development in other fungi. We found that each of these TFs is individually dispensable for room-temperature (RT) induced filamentation but each is required for other aspects of RT development. FBC1 and PAC2, but not STU1, are necessary for filamentation in response to cAMP at 37{degrees}C. Ectopic expression of each of these TFs is sufficient to induce filamentation at 37{degrees}C. Finally, PAC2 induction of filamentation at 37{degrees}C is dependent on STU1, suggesting these TFs form a regulatory circuit that, when activated at RT, promotes the hyphal program. ImportanceFungal illnesses pose a significant disease burden. However, the regulatory circuits that govern the development and virulence of fungi remain largely unknown. This study utilizes chemicals that can override the normal growth morphology of the human pathogen Histoplasma. Using transcriptomic approaches, we identify novel regulators of hyphal morphology and refine our understanding of the transcriptional circuits governing morphology in Histoplasma.

microbiology↗