Halabi, D. | bioRxiv Science

bioRxiv2026

Mismatch tolerance of a gRNA for CRISPR-based gene activation confers broad activity critical for cell reprogramming

CRISPR activation and interference systems (CRISPRa/i) are widely used for programmable transcriptional control. Although these technologies are capable of highly specific single-gene activity, some applications of transcriptional network reprogramming require broad, genome-wide effects. Here, we identify a CRISPRa gRNA that robustly reprograms astrocyte transcriptional state. Unexpectedly, this activity arises from extensive off-target binding that induces expression changes in thousands of genes, unlike neighboring gRNAs targeting the same intended on-target site. We leverage this promiscuous gRNA to dissect determinants of gRNA-driven off-target dCas9 binding in the context of transcriptional reprogramming. Using ChIP-seq, high-throughput protein-binding microarrays, and gRNA-variant library screening in cells, we demonstrate that PAM-proximal bases are primary determinants of genomic binding, mismatch tolerance is both gRNA- and base-specific, and targeted mutations within the PAM-proximal region can tune gRNA specificity. We further demonstrate that CRISPRa-driven phenotypes can reflect combined contributions from widespread off-target activity and dose-dependent on-target effects. These findings highlight the potentially widespread impacts of CRISPRa off-target activity, underscore the need to account for cryptic effects when selecting and evaluating gRNAs for programming cell phenotypes, and demonstrate that multi-site binding by CRISPRa systems can be exploited as a feature for network-level perturbations in cell reprogramming. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=83 SRC="FIGDIR/small/703129v1_ufig1.gif" ALT="Figure 1"> View larger version (34K): org.highwire.dtl.DTLVardef@b697b0org.highwire.dtl.DTLVardef@1a0b390org.highwire.dtl.DTLVardef@16ce710org.highwire.dtl.DTLVardef@b5d87a_HPS_FORMAT_FIGEXP M_FIG C_FIG

genomics↗

bioRxiv2025

Comprehensive profiling of transcription factors for reprogramming human astrocytes to neuronal cells through endogenous CRISPR-based gene activation

Neuronal loss is a hallmark of neurodegeneration and brain injury. Direct reprogramming of astrocytes into neurons has emerged as a promising approach to restore lost neurons. Comprehensive mapping and characterization of candidate astrocyte-to-neuron reprogramming factors is an essential step to realizing the potential of this strategy. Here, we established a CRISPR activation (CRISPRa)-based approach for neuronal reprogramming of primary human astrocytes. We conducted high-throughput CRISPRa screens of all human genes encoding transcription factors (TFs) to identify novel and efficient reprogramming factors. scRNA-seq characterization of top hits revealed that single TFs reprogram primary human astrocytes into multiple neuronal subtypes with distinct cell type-specific gene signatures. We demonstrate that INSM1 reprograms astrocytes to a glutamatergic neuron-like state and has broad neurogenic activity across different cell types and across human and mouse contexts. Finally, we conduct paired CRISPRa screens to identify cofactors that cooperate with INSM1 to enhance neuronal reprogramming and subtype specification, and elucidate genomic mechanisms of interaction and downstream regulators.

cell biology↗