Biotechnological applications of mobile group II introns and their reverse transcriptases: gene targeting, RNA-seq, and non-coding RNA analysisReport as inadecuate




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Mobile DNA

, 5:2

Mobile genetic elements in biotechnology

Abstract

Mobile group II introns are bacterial retrotransposons that combine the activities of an autocatalytic intron RNA a ribozyme and an intron-encoded reverse transcriptase to insert site-specifically into DNA. They recognize DNA target sites largely by base pairing of sequences within the intron RNA and achieve high DNA target specificity by using the ribozyme active site to couple correct base pairing to RNA-catalyzed intron integration. Algorithms have been developed to program the DNA target site specificity of several mobile group II introns, allowing them to be made into ‘targetrons.’ Targetrons function for gene targeting in a wide variety of bacteria and typically integrate at efficiencies high enough to be screened easily by colony PCR, without the need for selectable markers. Targetrons have found wide application in microbiological research, enabling gene targeting and genetic engineering of bacteria that had been intractable to other methods. Recently, a thermostable targetron has been developed for use in bacterial thermophiles, and new methods have been developed for using targetrons to position recombinase recognition sites, enabling large-scale genome-editing operations, such as deletions, inversions, insertions, and ‘cut-and-pastes’ that is, translocation of large DNA segments, in a wide range of bacteria at high efficiency. Using targetrons in eukaryotes presents challenges due to the difficulties of nuclear localization and sub-optimal magnesium concentrations, although supplementation with magnesium can increase integration efficiency, and directed evolution is being employed to overcome these barriers. Finally, spurred by new methods for expressing group II intron reverse transcriptases that yield large amounts of highly active protein, thermostable group II intron reverse transcriptases from bacterial thermophiles are being used as research tools for a variety of applications, including qRT-PCR and next-generation RNA sequencing RNA-seq. The high processivity and fidelity of group II intron reverse transcriptases along with their novel template-switching activity, which can directly link RNA-seq adaptor sequences to cDNAs during reverse transcription, open new approaches for RNA-seq and the identification and profiling of non-coding RNAs, with potentially wide applications in research and biotechnology.

KeywordsGenome engineering Metabolic engineering Next-generation RNA sequencing Ribozyme Synthetic biology Systems biology Targetron AbbreviationsCRACCross-linking and analysis of cDNA

DI-DVIGroup II intron RNA domains I-VI

DDNA-binding domain of group II intron reverse transcriptases

E1 and E25’ and 3’ exons

EBSExon-binding site

EnDNA endonuclease domain of group II intron reverse transcriptases

HITS-CLIPHigh-throughput sequencing by cross-linking immunoprecipitation

IBSIntron-binding site

I-ΔORFGroup II intron with ORF encoding the RT deleted

IEPIntron-encoded protein

PAMProtospacer adjacent motif

RAMRetrotransposition-activated marker

RIP-SeqRNA immunoprecipitation and sequencing

RNPRibonucleoprotein

RTReverse transcriptase

TpTrimethoprim.

Electronic supplementary materialThe online version of this article doi:10.1186-1759-8753-5-2 contains supplementary material, which is available to authorized users.

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Author: Peter J Enyeart - Georg Mohr - Andrew D Ellington - Alan M Lambowitz

Source: https://link.springer.com/







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