Helicase-assisted continuous editing for programmable mutagenesis of endogenous genomes.

Deciphering the context-specific relationship between sequence and function is a major challenge in genomics. Existing tools for inducing locus-specific hypermutation and evolution in the native genome context are limited. Here we present a programmable platform for long-range, locus-specific hypermutation called helicase-assisted continuous editing (HACE). HACE leverages CRISPR-Cas9 to target a processive helicase-deaminase fusion that incurs mutations across large (>1000-base pair) genomic intervals. We applied HACE to identify mutations in mitogen-activated protein kinase kinase 1 (MEK1) that confer kinase inhibitor resistance, to dissect the impact of individual variants in splicing factor 3B subunit 1 (SF3B1)-dependent missplicing, and to evaluate noncoding variants in a stimulation-dependent immune enhancer of CD69. HACE provides a powerful tool for investigating coding and noncoding variants, uncovering combinatorial sequence-to-function relationships, and evolving new biological functions.

Radiation dose enhancement using gold nanoparticles with a diamond linear accelerator target: a multiple cell type analysis.

Radiotherapy (RT) is an effective cancer treatment modality, but standard RT often causes collateral damage to nearby healthy tissues. To increase therapeutic ratio, radiosensitization via gold nanoparticles (GNPs) has been shown to be effective. One challenge is that megavoltage beams generated by clinical linear accelerators are poor initiators of the photoelectric effect. Previous computer models predicted that a diamond target beam (DTB) will yield 400% more low-energy photons, increasing the probability of interacting with GNPs to enhance the radiation dose by 7.7-fold in the GNP vicinity. After testing DTB radiation coupled with GNPs in multiple cell types, we demonstrate decreased head-and-neck cancer (HNC) cell viability in vitro and enhanced cell-killing in zebrafish xenografts compared to standard RT. HNC cell lines also displayed increased double-stranded DNA breaks with DTB irradiation in the presence of GNPs. This study presents preclinical responses to GNP-enhanced radiotherapy with the novel DTB, providing the first functional data to support the theoretical evidence for radiosensitization via GNPs in this context, and highlighting the potential of this approach to optimize the efficacy of RT in anatomically difficult-to-treat tumors.