Strategies and Advances in Site-Specific Integration of Exogenous Large Genes

Area of Science:

• Genetics
• Molecular Biology
• Biotechnology

Background:

• Large genomic deletions (≥1 kb) are a significant cause of monogenic disorders, often leading to gene inactivation or loss of regulatory elements.
• Current therapeutic strategies require methods for stable, targeted delivery of large DNA sequences with enhanced safety profiles.
• Existing methods like viral vectors and transposons often result in random integration, posing risks of insertional mutagenesis and variable transgene expression.

Purpose of the Study:

• To review and synthesize recent advancements in targeted large-fragment DNA integration technologies.
• To compare different locus-specific strategies for their potential in genetic therapeutics and disease modeling.
• To provide a perspective on the performance, limitations, and future applications of these emerging technologies.

Main Methods:

• Review of homology-directed repair (HDR)-dependent CRISPR-Cas9 strategies for kilobase-scale DNA payloads.
• Examination of prime editing approaches, including those combined with engineered recombinases for large DNA cargo insertion.
• Survey of emerging HDR-independent systems utilizing CRISPR-guided transposition and retrotransposition.

Main Results:

• Genome editing has diversified, offering a growing repertoire of locus-specific strategies for large-fragment DNA insertion.
• HDR-dependent CRISPR-Cas9, prime editing, and CRISPR-guided transposition/retrotransposition represent key classes of targeted integration technologies.
• These methods offer improved control over insertion site and copy number compared to legacy approaches.

Conclusions:

• Targeted large-fragment integration technologies are revolutionizing genetic therapeutics and disease model construction.
• Ongoing advancements in genome editing provide precise and safer alternatives for correcting large genomic defects.
• These technologies hold significant promise for broader biomedical applications in treating genetic disorders.