Biology subjects

Wasko, K. M.

Publications and source records attributed to Wasko, K. M..

5 recordsLinked to original sources

bioRxiv2026

Programming T cells for intercellular genome editing

Therapeutic genome editing requires delivery of editing molecules to defined cell types, but targeting specificity and efficiency are currently limited. We hypothesized that properties inherent to immune cells, including tissue infiltration and programmed cell recognition, could be harnessed to engineer a cell-based delivery system. We show here that T cells can both produce and transfer editing machinery to target cells. In response to a programmable ligand, engineered T-lymphoid cells can transfer enzymes using complex spatiotemporal logic and deliver cargo in a cell contact-dependent or -independent manner. We demonstrate feasibility of this approach in primary human T cells, establishing a customizable genetic circuit for macromolecular delivery controlled by intercellular interactions.

bioengineering↗

bioRxiv2026

Spligation enables programmable chimeric RNA generation in living cells

The ability to precisely modify RNA offers opportunities to manipulate the flow of genetic information and influence transcript stability, localization and translation. RNA-targeting technologies enable RNA knockdown, base editing and trans-splicing, but more extensive transcript changes typically require genome editing or rely on the endogenous splicing machinery. Based on the ability of type III-A CRISPR-Csm complexes to catalyze programmable RNA cleavage in human cells, we investigated their potential to induce site-specific deletions while leaving the remainder of the transcript intact. Our data show that CRISPR-Csm complexes can generate short and long RNA excisions within a target transcript, and that the efficiency of this process is enhanced by fusion of Csm to the RNA ligase RtcB. Furthermore, cleavage of two different transcripts can trigger subsequent trans-ligation of the cleaved products into a chimeric transcript ("spligation"). Finally, we apply spligation to endogenous transcripts, using Csm to generate recombinant mRNA in cells independent of canonical splice sites. Collectively, this approach enables new forms of precise RNA manipulation in cells with potential applications in human disease.

bioengineering↗

bioRxiv2026

Amplified genome editing by in vivo editor production

Genome editing enzymes have vast therapeutic potential. However, achieving sufficient delivery in vivo remains a major challenge, because editing machinery is confined to the subset of transfectable cells in a tissue. Here, we tested the possibility that genome editing could be amplified in vivo by enabling transfected cells to transfer editing enzymes to neighboring cells. Our data show that this NANoparticle-Induced Transfer of Enzyme (NANITE) strategy quadrupled editing efficiency in cultured cells relative to non-spreading controls. A single intravenous injection of the NANITE plasmid into mice induced ~3-fold higher levels of liver editing at the transthyretin (Ttr) locus relative to non-spreading controls, with corresponding reductions in serum TTR levels. Spreading therapeutic enzymes offers a nonviral and non-infectious strategy to boost therapeutic effects after delivery.

bioengineering↗

bioRxiv2026

Stepwise DNA unwinding gates TnpB genome-editing activity

TnpB is a compact RNA-guided endonuclease and evolutionary ancestor of CRISPR-Cas12 that offers a promising platform for genome engineering. However, the genome-editing activity of TnpBs remains limited and its underlying determinants are poorly understood. Here, we used biochemical and single-molecule assays to examine the DNA-unwinding mechanism of Youngiibacter multivorans TnpB (Ymu1 TnpB). DNA unwinding proceeds through a discrete, long-lived partially unwound intermediate state before reaching a fully unwound open state. The open state forms inefficiently and collapses readily in the absence of negative supercoiling. An optimized variant, Ymu1-WFR, stabilizes formation of both the intermediate and open states, resulting in enhanced DNA cleavage in vitro and increased genome editing in plants. These findings identify the physical basis for the observed minimal activities of natural TnpBs, revealing how stabilizing specific unwinding states enables efficient DNA targeting.

biochemistry↗

bioRxiv2025

Directed evolution expands CRISPR-Cas12a genome editing capacity

CRISPR-Cas12a enzymes are versatile RNA-guided genome-editing tools with applications encompassing viral diagnosis, agriculture and human therapeutics. However, their dependence on a 5-TTTV-3 protospacer-adjacent motif (PAM) next to DNA target sequences restricts Cas12as gene targeting capability to only [~]1% of a typical genome. To mitigate this constraint, we used a bacterial-based directed evolution assay combined with rational engineering to identify variants of Lachnospiraceae bacterium Cas12a (LbCas12a) with expanded PAM recognition. The resulting Cas12a variants use a range of non-canonical PAMs while retaining recognition of the canonical 5-TTTV-3 PAM. In particular, biochemical and cell-based assays show that the variant Flex-Cas12a utilizes 5-NYHV-3 PAMs that expand DNA recognition sites to [~]25% of the human genome. With enhanced targeting versatility, Flex-Cas12a unlocks access to previously inaccessible genomic loci, providing new opportunities for both therapeutic and agricultural genome engineering.

biochemistry↗