Corden, E. | Search bioRxiv

bioRxiv2026

Quantitative profiling of intrinsic dCas9-DNA recognition reveals key determinants of guide RNA performance

CRISPR technologies based on nuclease-deactivated Cas9 (dCas9) rely on programmable DNA binding rather than DNA cleavage, yet the intrinsic DNA-recognition properties that govern optimal guide RNA (gRNA) performance remain poorly understood. Existing approaches either measure genomic occupancy in cells or infer dCas9 behavior from cleavage-based Cas9 datasets, despite DNA binding being substantially more permissive than DNA cleavage. Here we introduce TANGO (Targeted Array-based Nucleic acid-Guided Occupancy), a high-density DNA-array platform that quantitatively profiles intrinsic dCas9:gRNA binding across tens of thousands of DNA targets in a cell-free system. TANGO captures established features of dCas9 target recognition, while providing substantially greater sensitivity than prior assays. Comparison with ChIP-seq data demonstrates that intrinsic DNA-binding specificity is a major driver of genomic occupancy and reveals that chromatin accessibility modulates the intrinsic binding affinity required for dCas9 recruitment. Across CRISPRi/a guides, TANGO identifies multiple independent biochemical determinants of guide performance--including on-target affinity, mismatch tolerance, and ribonucleoprotein assembly--and flags problematic and highly promiscuous guides overlooked by current specificity metrics. Unexpectedly, some guides retain substantial guide-directed DNA binding even in the absence of a protospacer-adjacent motif (PAM), revealing an additional dimension of dCas9 specificity. Together, these results establish intrinsic DNA recognition as a quantitative and experimentally accessible determinant of dCas9 function, providing a framework for improving guide selection and enhancing the precision of CRISPR technologies.

genomics↗

bioRxiv2024

TFAM as a sensor of UVC-induced mitochondrial DNA damage

Mitochondria lack nucleotide excision repair; however, mitochondrial DNA (mtDNA) is resistant to mutation accumulation following DNA damage. These observations suggest additional damage sensing or protection mechanisms. Transcription Factor A, Mitochondrial (TFAM) compacts mtDNA into nucleoids and binds differentially to certain forms of DNA damage. As such, TFAM has emerged as a candidate for protecting mtDNA or sensing damage. To examine the possibilities that TFAM might protect DNA from damage or act as a damage sensing protein for irreparable forms of mtDNA damage, we used live-cell imaging and HeLa cell-based assays, atomic force microscopy (AFM), and high-throughput protein-DNA binding assays to characterize the binding properties of human TFAM to ultraviolet-C (UVC) irradiated DNA and the cellular consequences of UVC irradiation. Our cell data show increased TFAM mRNA after exposure and suggest an increase in mtDNA degradation without a loss in mitochondrial membrane potential that might trigger mitophagy. Our protein-DNA binding assays indicate a reduction in sequence specificity of TFAM following UVC irradiation and a redistribution of TFAM binding throughout the mitochondrial genome. Our AFM data show increased compaction of DNA by TFAM in the presence of damage. Despite the TFAM-mediated compaction of mtDNA in vitro, we do not observe any protective effect of increased TFAM protein on DNA damage formation in cells or in vitro. Increased TFAM protein did not alter levels of mtDNA damage over time after UVC exposure in vivo, but knockdown of TFAM did alter mtDNA damage levels in HeLa cells both at baseline and after UVC exposure. Taken together, these studies indicate that UVC-induced DNA damage alters TFAM binding and promotes compaction by TFAM in vitro. We hypothesize that that TFAM may act as a damage sensing protein in vivo, sequestering damaged genomes to prevent mutagenesis by facilitating removal or suppression of replication.

biochemistry↗