Mobile Group II Introns: Site‐Specific DNA Integration and Applications in Gene Targeting

Abstract

Mobile group II introns are bacterial retroelements that integrate site-specifically into new DNA target sites in a process called “retrohoming”. Retrohoming is mediated by an RNP that is formed during RNA splicing and consists of the catalytically active intron RNA (“ribozyme”) and an intron-encoded reverse transcriptase (RT). RNPs recognize DNA target sequences for retrohoming by using both the protein and base pairing of the intron RNA, with the latter contributing most of the DNA target specificity. Site-specific DNA integration then ensues by a remarkable mechanism in which the intron RNA uses its ribozyme activity to insert (“reverse splice”) directly into a DNA strand, where it is reverse transcribed back into DNA by the intron-encoded RT. Because the DNA target site is recognized largely by base pairing of the intron RNA with little contribution from the protein, the intron can be reprogrammed to insert into new DNA target sites simply by modifying the intron RNA. This feature combined with their high insertion frequencies and specificity enabled the development of mobile group II introns into gene targeting vectors (“targetrons”), with the unique feature of readily programmable DNA target specificity. Targetrons are now widely used for genetic engineering and functional genomics in Gram-negative and Gram-positive bacteria and are being adapted for use in higher organisms.