Group II Introns as Gene Targeting Vectors

The novel DNA insertion mechanism used by group II introns has enabled the development of a new technology for manipulating DNA. Group II introns adapted for gene targeting have been dubbed "targetrons". The introns have a number of characteristics that make them well suited for gene targeting. First, they have very high insertion frequencies, which can approach 100%. Second, they have long (-30 bp) target sequences, which result in high specificity. Third, the introns can be programmed to insert into new target sequences simply by modifying the RNA sequences that pair with the DNA target. Fourth, foreign sequences can be inserted within the loop of domain IV and then delivered site-specifically with the intron. Finally, because the introns are largely self-contained and require only commonly available DNA recombination or repair enzymes to complete cDNA integration, they have a wide hostrange and can potentially function in any organism.

In bacteria, targetrons are expressed from a donor plasmid, such as pACD3, which is used for targetron expression in E. coli (Fig. 6; Karberg et al. 2001; Zhong et al. 2003). pACD3 employs an inducible T7lac promoter to express an Ll.LtrB-AORF intron and short flanking exons, with the IEP expressed from a position just downstream of the 3' exon. The IEP expressed from this position promotes efficient splicing of the intron RNA, forming the RNP homing endonuclease that promotes mobility, but when the intron inserts into a new location, it does not carry the ORF, and thus is unable to splice. The use of the AORF intron also greatly increases the insertion frequency by making the intron RNA less susceptible to nuclease digestion (Guo et al. 2000). Analogous vectors have been developed for the LLLtrB targetron in other bacteria with appropriate modifications of the promoter and Shine-Dalgarno sequence used for intron RNA and IEP expression (e.g. Frazier et al. 2003), and the same approaches should be readily adaptable for other group II introns.

The intron is targeted to specific sites with the help of a computer algorithm that scans the target sequence for the best matches to the positions recognized by the IEP and then designs primers to modify the intron's EBS and 6 sequences to insert into those sites (Perutka et al. 2004). The positions recognized by the IEP are sufficiently few and flexible that the program readily identifies multiple rank-ordered target sites in any gene. The IBS sequences in the 5' exon of the precursor RNA must also be made complementary to the retargeted EBS sequences for efficient RNA splicing. The required modifications are introduced into the donor plasmid via polymerase chain reaction (PCR; Zhong et al. 2003). In E. coli, targetrons commonly insert in the desired chro-

Protein Splicing Images

Fig. 6. Use of targetrons for gene disruption. The targetron donor plasmid (pACD3) uses a T7lac promoter to express a AORF-derivative of the Ll.LtrB intron and short flanking exons, with the IEP expressed from a position just downstream of the 3' exon. Introns targeted to sites in the top (sense) or bottom (antisense) strands insert in different orientations relative to target gene transcription. A AORF intron inserted in the sense orientation can potentially yield a conditional disruption by linking its splicing to the expression of the IEP from a separate construct (Karberg et al. 2001; Frazier et al. 2003). An intron inserted in the antisense orientation cannot be spliced, yielding an unconditional disruption. Pc Promoter of targeted chromosomal gene

Fig. 6. Use of targetrons for gene disruption. The targetron donor plasmid (pACD3) uses a T7lac promoter to express a AORF-derivative of the Ll.LtrB intron and short flanking exons, with the IEP expressed from a position just downstream of the 3' exon. Introns targeted to sites in the top (sense) or bottom (antisense) strands insert in different orientations relative to target gene transcription. A AORF intron inserted in the sense orientation can potentially yield a conditional disruption by linking its splicing to the expression of the IEP from a separate construct (Karberg et al. 2001; Frazier et al. 2003). An intron inserted in the antisense orientation cannot be spliced, yielding an unconditional disruption. Pc Promoter of targeted chromosomal gene mosomal target sites at frequencies >1% without selection, readily detectable by colony PCR screening (Perutka et al. 2004).

Targetrons containing conventional or retrotransposition-activated selectable markers (RAM) inserted in DIV can be used to genetically select intron-integration events. RAM markers are patterned after previously described retrotransposition-indicator gene (RIG) markers (Ichiyanagi et al. 2002). The prototype RAM marker used for gene targeting was a small trimethoprim-re-sistance (TpR) gene inserted in group II intron DIV in the reverse orientation, but containing an efficiently self-splicing group I intron (the phage T4 td intron) inserted in the forward orientation. The group I intron is excised during retrotransposition, activating the TpR gene (Zhong et al. 2003). Typically, nearly 100% of the TpR colonies have correctly targeted introns, with few if any having non-specific insertions. A kanR-RAM marker has also been used for gene targeting (unpubl. data), and, in principle, the same approach could be used for any other selectable or screenable marker.

Gene Disruption by Insertional Mutagenesis. The Ll.LtrB targetron has been used for targeted gene disruption in E. coli and other Gram-negative and Gram-positive bacteria (Karberg et al. 2001; Frazier et al. 2003). Because the targetron contains multiple stop codons in all reading frames, suitably placed disruptions totally ablate gene function. The intron RNA can be targeted to insert in either strand, resulting in different orientations relative to target gene transcription. An intron that inserts in the antisense orientation cannot be spliced and gives an unconditional disruption, whereas an intron that inserts in the sense orientation can potentially yield a conditional disruption by linking its splicing to the expression of the IEP from a separate construct containing an inducible promoter (Fig. 6; Karberg et al. 2001; Frazier et al. 2003). In practice, the splicing efficiency of the retargeted intron is generally less than that of the wild-type intron, likely due to a combination of suboptimal exon contexts at new target sites and decreased splicing efficiency when the IEP is expressed in trans. Consequently, conditional disruptions can be obtained in some cases but not others. This situation may be improved by optimizing the IEP for expression in trans.

By incorporating a RAM marker, an LLLtrB intron with randomized target site recognition sequences was used to obtain disruptions at sites distributed throughout the E. coli genome, analogous to global transposon mutagenesis (Zhong et al. 2003). Despite clustering of insertions near the chromosome replication origin, the resulting library was sufficiently complex to contain most viable E. coli gene disruptions. Recent studies on Ll.LtrB retrotransposition suggest that altering the growth conditions may lead to a more uniform distribution of insertion sites (Coros et al. 2005). In addition, group II introns that are inserted into any gene can be "fished" from the library by PCR and inserted into a donor plasmid to obtain single disruptions in the same or different host strains, an important advantage over libraries generated with conventional transposons (Zhong et al. 2003; J. Yao, J. Zhong, A.M. Lambowitz, in prep.). The incorporation of a RAM marker also makes it possible to automate whole genome library construction by designing introns targeted to each gene and using robots to select antibiotic-resistant colonies. The advantages are that each gene can be targeted individually, with multiple introns if necessary, and that high insertion frequencies in the absence of a selectable marker facilitate the construction of strains having multiple disruptions or other desirable combinations of traits.

Site-Specific DNA Insertion. In addition to disrupting genes, group II introns can be used to integrate cargo genes inserted in DIV at desired chromosomal locations (e.g., Frazier et al. 2003). The insertion of extra DNA in DIV may decrease the integration frequency by making the intron more susceptible to degradation by host nucleases (see Guo et al. 2000). In most cases, however, the frequency remains sufficiently high to detect the desired integration. In the example shown in Fig. 7, this approach was used to engineer lactobac-teria by inserting a commercially important phage resistance gene (abiD) into a specific genomic locus, thereby conferring regulatable phage resistance. The desired insertion was identified at a frequency of 0.5-2% by colony PCR screening. The ability to obtain desired insertions without selection for antibiotic resistance is particularly important in food-grade organisms like lacto-bacteria.

Targeted Double-Strand Breaks. Experiments with rare-cutting DNA endo-nucleases have shown that the introduction of double-strand breaks in bacterial, plant, or animal chromosomal DNA greatly stimulates homologous recombination with a cotransformed DNA fragment, enabling the introduction of desired mutations by gene replacement (Jasin 1996; Donoho et al. 1998). However, despite efforts at protein engineering (Chevalier et al. 2002; Epinat et al. 2003), the power of this approach remains limited by the fixed specificity of protein endonucleases. Group II introns can be used similarly, with the advantage that the double-strand break can be targeted to any desired chromosome region. The proof-of-principle experiment shown in Fig. 8 used an Ll.LtrB intron with a point mutation abolishing the RT activity of the IEP to prevent cDNA synthesis at the cleaved target site. The RT-deficient intron was targeted to introduce a double-strand break (reverse splicing plus second-strand cleavage) in the E. coli chromosomal thyA gene, which was then repaired by homologous recombination with a cotransformed plasmid containing a segment of the thyA gene with stop codons at desired locations (Karberg et al. 2001). In the absence of cDNA synthesis, the reverse-spliced intron RNA was presuma-

Targetron Donor

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3, Site-Specific Chromosomal 5 Insertion

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3, Site-Specific Chromosomal 5 Insertion

Fig. 7. Use of targetrons for site-specific insertion of a cargo gene at a desired genomic location. In the example shown, the abiD gene, which confers phage resistance, was inserted at a site within the regulata-ble mleS gene in the L. Iactis chromosome (Frazier et al. 2003). An Ll.LtrB-AORF intron targeted to the mleS gene with the abiD gene inserted in intron DIV in place of the IEP was expressed from an L. lactis donor plas-mid using a nisin-inducible promoter (PNis)- The intron is flanked by short exon sequences (El and E2). The IEP, which splices and mobilizes the intron, is cloned downstream of E2. Splicing results in the formation of an RNP containing the IEP and intron lariat RNA with the abiD gene in DIV, which is then inserted into the chromosomal DNA target site by retrohoming bly degraded by cellular enzymes, leaving a double-strand break that could be acted on by cellular enzymes. The introduction of the double-strand break by targeted LLLtrB introns stimulated homologous recombination frequencies in E. coli by one to two orders of magnitude (Karberg et al. 2001).

Use of Targeted Group II Introns in Eukaryotes. If appropriate methods can be developed for eukaryotes, mobile group II introns would have potentially wide medical, agricultural, and commercial applications, including the introduction of genetically stable gene disruptions for functional genomics, and the site-specific introduction or repair of genes for genetic engineering and gene therapy. In initial work establishing the potential of the approach, group II introns were targeted to insert into the HIV-1 provirus and the human gene encoding CCR5, an important target site in anti-HIV-1 therapy (Guo et al. 2000). The retargeted introns were shown to insert at high frequencies in-

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Chromosome "

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Fig. 8. Use of targetrons to introduce targeted doublestrand breaks that stimulate homologous recombination with a cotransformed DNA fragment. In the example shown, an Ll.LtrB intron targeted to the E. coli thyA gene was used to introduce stop codons into the thyA gene in the E. coli chromosome (Karberg et al. 2001). The intron carrying an RT~ mutation to prevent cDNA synthesis at the target site was expressed from a donor plasmid (not shown) to produce an RNP that makes a double-strand break at the chromosomal thyA target site (arrow). The double-strand break then stimulates homologous recombination with a cotransformed plasmid (pBRR-ThyA-Stop) carrying a segment of the thyA gene with stop codons at desired sites (black boxes)

to their desired target sites in an E. coli plasmid assay, and the intron RNPs retained activity in human cells inserting into a plasmid target site in a lipo-some-mediated transfection assay. Current work focuses on developing more efficient methods for introducing or expressing RNPs in mammalian cells, in order to obtain a sufficiently high concentration of RNPs in the nucleus for efficient chromosome integration.

Acknowledgements. Work in the author's laboratories was supported by NIH grants GM37949 and GM3791 to A.M.L. and CIHR, NSERC, and AHFMR grants to S.Z.

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