Large genomic deletions (≥1 kb) are a recurrent class of disease-causing lesions in monogenic disorders, frequently leading to complete gene inactivation or the loss of critical cis-regulatory elements. Addressing these defects in a therapeutically relevant manner requires integration modalities capable of delivering and stably installing large exogenous DNA sequences at predefined genomic loci with an improved safety profile. By contrast, legacy approaches-including viral-vector delivery, recombinase-based strategies, and transposon-mediated insertion-typically achieve integration through random or semi-random mechanisms, which, despite their practicality and often favorable efficiencies, limit control over insertion site and copy number and may increase the risk of insertional mutagenesis and position-dependent variability in transgene expression. The past few years have witnessed rapid methodological diversification driven by genome editing, resulting in a growing repertoire of locus-specific strategies for large-fragment DNA insertion that are reshaping both disease-model construction and genetic therapeutics. In this Review, we synthesize the main classes of targeted large-fragment integration technologies reported to date. We begin with homology-directed repair (HDR)-dependent CRISPR-Cas9 knock-in strategies and discuss how donor architecture and local donor recruitment can be leveraged to improve integration outcomes for kilobase-scale payloads. We then examine approaches centered on prime editing, particularly those that couple prime editing with engineered serine/tyrosine recombinases to support programmable insertion of large DNA cargos. We close by surveying emerging HDR-independent systems based on CRISPR-guided transposition and retrotransposition, and we provide a comparative perspective on their performance envelopes, constraints, and trajectories toward broader biomedical applications.
