Precise cut-and-paste DNA insertion using engineered type V-K CRISPR-associated transposases.

CRISPR-associated transposases (CASTs) enable recombination-independent, multi-kilobase DNA insertions at RNA-programmed genomic locations. However, the utility of type V-K CASTs is hindered by high off-target integration and a transposition mechanism that results in a mixture of desired simple cargo insertions and undesired plasmid cointegrate products. Here we overcome both limitations by engineering new CASTs with improved integration product purity and genome-wide specificity. To do so, we engineered a nicking homing endonuclease fusion to TnsB (named HELIX) to restore the 5' nicking capability needed for cargo excision on the DNA donor. HELIX enables cut-and-paste DNA insertion with up to 99.4% simple insertion product purity, while retaining robust integration efficiencies on genomic targets. HELIX has substantially higher on-target specificity than canonical CASTs, and we identify several novel factors that further regulate targeted and genome-wide integration. Finally, we extend HELIX to other type V-K orthologs and demonstrate the feasibility of HELIX-mediated integration in human cell contexts.

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.

The identification of dual protective agents against cisplatin-induced oto- and nephrotoxicity using the zebrafish model.

Dose-limiting toxicities for cisplatin administration, including ototoxicity and nephrotoxicity, impact the clinical utility of this effective chemotherapy agent and lead to lifelong complications, particularly in pediatric cancer survivors. Using a two-pronged drug screen employing the zebrafish lateral line as an in vivo readout for ototoxicity and kidney cell-based nephrotoxicity assay, we screened 1280 compounds and identified 22 that were both oto- and nephroprotective. Of these, dopamine and L-mimosine, a plant-based amino acid active in the dopamine pathway, were further investigated. Dopamine and L-mimosine protected the hair cells in the zebrafish otic vesicle from cisplatin-induced damage and preserved zebrafish larval glomerular filtration. Importantly, these compounds did not abrogate the cytotoxic effects of cisplatin on human cancer cells. This study provides insights into the mechanisms underlying cisplatin-induced oto- and nephrotoxicity and compelling preclinical evidence for the potential utility of dopamine and L-mimosine in the safer administration of cisplatin.