Gene targeting using randomly inserted group II introns (targetrons) recovered from an Escherichia coli gene disruption library.
The Lactococcus lactis Ll.LtrB group II intron retrohomes by reverse-splicing into one strand of a double-stranded DNA target site, while the intron-encoded protein cleaves the opposite strand and uses it to prime reverse transcription of the inserted intron RNA. The protein and intron RNA function in a ribonucleoprotein particle, with much of the DNA target sequence recognized by base-pairing of the intron RNA. Consequently, group II introns can be reprogrammed to insert into specific or random DNA sites by substituting specific or random nucleotide residues in the intron RNA. Here, we show that an Escherichia coli gene disruption library obtained using such randomly inserting Ll.LtrB introns contains most viable E.coli gene disruptions. Further, each inserted intron is targeted to a specific site by its unique base-pairing regions, and in most cases, could be recovered by PCR and used unmodified to obtain the desired single disruptant. Additionally, we identified a subset of introns that insert at sites lacking T+5, a nucleotide residue critical for second-strand cleavage. All such introns tested individually gave the desired specific disruption, some by switching to an alternate retrohoming mechanism targeting single-stranded DNA and using a nascent lagging DNA strand to prime reverse transcription.
Yao
Jun
J
Institute for Cellular and Molecular Biology, Department of Chemistry and Biochemistry, and Section of Molecular Genetics and Microbiology, School of Biological Sciences, University of Texas at Austin Austin, TX 78712, USA.
Zhong
Jin
J
Lambowitz
Alan M
AM
D020029:Base Pairing; D004247:DNA / Q000737:chemistry; D004926:Escherichia coli / Q000235:genetics*; D015723:Gene Library; D018390:Gene Targeting / Q000379:methods*; D005798:Genes, Bacterial; D007438:Introns; D013294:Lactococcus lactis / Q000235:genetics; D010957:Plasmids; D016133:Polymerase Chain Reaction; D018626:Retroelements
D018626:Retroelements; D004247:DNA