Fourth-generation gene editors: Integration-based genome engineering

Gene editing has rapidly progressed from early methods into highly precise tools with immense therapeutic promise. While initial CRISPR-Cas9 systems revolutionized the field, the fact that they rely on double-strand breaks can lead to genomic instability and off-target effects, which calls for innovation. This review covers the advancements in fourth-generation gene editing technologies. Specifically, it focuses on diverse integrase systems that enable precise DSB-free DNA insertion. First, we explore site-specific recombinases like tyrosine recombinases (Cre, FLP), large serine recombinases (PhiC31, Bxb1), and bridge recombinases (IS622) that offer efficient DNA rearrangements. Next, we introduce DNA transposases, which cover a versatile cut and paste systems (piggyBac, Sleeping Beauty), as well as the CRISPR-associated transposons (CASTs, type I-F, type V-K), which achieve RNA-guided, DSB-free DNA insertion with high efficiency and directionality. This review also highlights viral integrases (HIV, MLV), known for their stable gene delivery, and mobile group II introns (targetrons) and R2 retrotransposons for their site-specific integration and all-RNA delivery strengths. Even though there have been significant advances in efficiency, specificity, and delivery strategies, challenges with these powerful tools remain. Continued refinement of these integrase systems will facilitate a new generation in gene-based approaches for the definitive correction of variants of inherited diseases.