Human gene therapyApr 24, 2026

Methods and Progress in Inserting Large External Genes into Specific DNA Sites

CRISPR Gene Editing Weekly Brief ↗PubMed ↗DOI ↗

Updated Apr 25, 2026

Abstract

Large genomic deletions (≥1 kb) are frequently associated with gene inactivation in monogenic disorders.

Integration modalities are needed to deliver and install large DNA sequences at specific genomic locations with improved safety.
Legacy integration approaches, while practical, can lead to random insertion and increased risk of unwanted mutations.
Recent advancements in genome editing have led to targeted strategies for inserting large DNA fragments, enhancing disease modeling and genetic therapies.
Homology-directed repair (HDR) methods utilizing CRISPR-Cas9 can improve integration outcomes for large DNA payloads by optimizing donor design.
Prime editing combined with engineered recombinases offers a programmable method for inserting large DNA segments.
Emerging HDR-independent systems based on CRISPR-guided transposition show potential for broader biomedical applications.

Simplified

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.

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Methods and Progress in Inserting Large External Genes into Specific DNA Sites

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