Delano, J. | Search bioRxiv

bioRxiv2024

CRISPR-CLEAR: Nucleotide-Resolution Mapping of Regulatory Elements via Allelic Readout of Tiled Base Editing

CRISPR tiling screens have advanced the identification and characterization of regulatory sequences but are limited by low resolution arising from the indirect readout of editing via guide RNA sequencing. This study introduces CRISPR-CLEAR, an end-to-end experimental assay and computational pipeline, which leverages targeted sequencing of CRISPR-introduced alleles at the endogenous target locus following dense base-editing mutagenesis. This approach enables the dissection of regulatory elements at nucleotide resolution, facilitating a direct assessment of genotype-phenotype effects.

genomics↗

bioRxiv2022

Biomolecular condensates govern PARP inhibitor trapping and present mechanisms of resistance

Poly(ADP-ribose) polymerase (PARP) inhibitors (PARPi) are a class of cancer drugs that enzymatically inhibit PARP activity at sites of DNA damage. In the context of BRCA mutations, PARPi can be synthetically lethal, presenting ideal genetic targeting. Yet, PARPi function primarily by trapping PARP1 onto sites of DNA damage. How PARPi trap and why some are better trappers remain unknown. Here, we show trapping occurs primarily through a kinetic phenomenon within biomolecular condensates that correlates with PARPi koff. Our results suggest PARP trapping is not the physical stalling of PARP1 on DNA, rather the high probability of PARP re-binding damaged DNA in the absence of other DNA binding protein recruitment. Furthermore, we found recruitment of the DNA binding protein RPA1 correlates to cell line PARPi sensitivity independent of RPA1 expression, demonstrating that condensate recruitment alone can impact efficacy. These results shed new light on how PARPi function, describe how PARPi properties correlate to trapping potency, and suggest previously unknown mechanisms of PARPi resistance.

biophysics↗