Engineering RNA-guided bridge recombinases for precise large-scale genome editing.

CRISPR-Associated Transposases Enable Programmable DNA Integration in Plants

Programmable DNA integration is a major challenge in plant genome engineering. CRISPR-associated transposases (CAST) catalyze efficient RNA-guided DNA integration without double-strand breaks, yet their activity has not been established in plants. Here, we reconstituted and engineered a Type I-F CAST for programmable DNA integration in plant cells. We validated expression of the wild-type Pseudoalteromonas CAST (PseCAST) machinery in plants and established targeted episomal integration in Arabidopsis thaliana protoplasts and chromosomal integration at a transgenic locus in Nicotiana benthamiana. The evolved PseCAST system, evoCAST, showed chromosomal integration efficiencies of 2.7%, representing a 6-fold improvement over wild-type PseCAST. evoCAST also enabled the insertion of cis-regulatory elements into a synthetic landing pad with 8% efficiency. evoCAST was subsequently retargeted to six endogenous genomic loci, demonstrating programmable integration across diverse chromosomal contexts. Finally, a cofactor screen identified the chromatin-associated factor AtHMGB2 as an enhancer of evoCAST-mediated integration activity in plants. These results establish CAST as a functional platform for programmable DNA insertion in plants and provide a foundation for developing targeted genome-engineering technologies for crop biotechnology.

Strategies and mechanisms of precision genome engineering: From gene editing to genome writing

Genomic manipulation has advanced from stochastic nuclease‐mediated disruption toward programmable, deterministic precision. Early clustered regularly interspaced short palindromic repeats (CRISPR) strategies enabled targeted mutagenesis through double‐strand breaks; however, their therapeutic application is limited by genotoxicity, chromosomal instability, and dependence on endogenous repair pathways that are difficult to predict. In this review, we examined the transition from gene editing to genome writing, an approach that decouples genomic modification from host repair pathways to better balance efficiency, precision, and payload delivery. We also discussed the principles of precision technologies, including base and prime editors, and described emerging large‐scale writers, such as CRISPR‐associated transposases and recombinase‐based bridge RNAs, which enable the integration of multi‐kilobase synthetic modules. Beyond enzymatic mechanisms, we further considered the combined use of generative artificial intelligence, structural biology, and novel delivery architectures as potential strategies to overcome current biological limitations. Taken together, these developments point toward Generative Biology, in which computational design and high‐throughput screening transform the genome from a static substrate into a more dynamic model for complex, synthetic functional design.

Efficient and precise programmable DNA knock-in without double-strand breaks.

Large-scale genome structure interrogation via recombinase-mediated rearrangements of multiplexed prime edits in repetitive elements.

This Protocol leverages prime editing to insert recombinase recognition sites into repetitive genomic regions, such as LINE-1 elements, thereby enabling extensive genetic modifications in human cells, and supports a wide range of studies, including genome-wide functional analyses and essentiality mapping.

Precise genomic integration of large DNA fragments by donor-directed annealing using prime editing.

A programmable gene replacement tool, named prime assembly (PA), which adapts prime editors to produce one or two pairs of 3'-flaps on both the genome and donor DNA, allowing megabase-scale genomic excision and/or kilobase-scale donor insertion at the gene of interest.

A Golden Gate-Compatible CRISPR-Associated Transposon Tool for Multiplexed Bacterial Genome Editing.