Published in: Science, vol. 289, no. 5478, pp. 452-457 (July 21, 2000): 

"Group II Introns Designed to Insert Into Therapeutically Relevant DNA Target Sites in Human Cells". 

Huatao Guo 1, Michael Karberg 1, Meredith Long 2, J. P. Jones III 2, Bruce Sullenger 2, and Alan M. Lambowitz 1*.

1 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, Austin, TX 78712, USA.
2 Center for Genetic and Cellular Therapies, Department of Surgery, Duke University Medical Center, Box 2601, Durham, NC 27710 USA.

* To whom correspondence should be addressed.
E-mail: lambowitz@mail.utexas.edu 


Abstract:
Introduction:
Fig. 1A:
Fig. 1B:
Fig. 1C:
References and Notes:
Additional Reference:
Links:
Feedback:

Abstract:

Mobile group II intron RNAs insert directly into DNA target sites and are then reverse-transcribed into genomic DNA by the associated intron-encoded protein. Target site recognition involves modifiable base-pairing interactions between the intron RNA and a >14-nucleotide region of the DNA target site, as well as fixed interactions between the protein and flanking regions. Here, we developed a highly efficient Escherichia coli genetic assay to determine detailed target site recognition rules for the Lactococcus lactis group II intron L1.LtrB and to select introns that insert into desired target sites. Using human immuno-deficiency virus-type 1(HIV-1) proviral DNA and the human CCR5 gene as examples, we show that group II introns can be retargeted to insert efficiently into virtually any target DNA and that the retargeted introns retain activity in human cells. This work provides the practical basis for potential applications of targeted group II introns in genetic engineering, functional genomics, and gene therapy. 


Introduction:

Group II introns are catalytic RNAs that function as mobile genetic elements by inserting directly into target sites in double-stranded DNA ( 1, 2). This mobility is mediated by a multi-functional intron-encoded protein (IEP) that has reverse transcriptase (RT), RNA splicing (maturase), and DNA endonuclease activities (2-5). After translation, the protein promotes RNA splicing, presumably by facilitating formation of the catalytically active intron RNA structure. It then remains associated with the excised intron to form a ribonucleoprotein (RNP) complex, which has DNA endonuclease/integrase activity. In homing, the major mobility pathway, the excised intron RNA in this complex reverse-splices into a specific target site in double-stranded DNA (6-8). The associated IEP then cleaves the opposite strand in the 3' exon of the DNA target, 9 or 10 nucleotides (nt) downstream of the intron insertion site, and uses the 3' end of the cleaved strand as a primer to reverse-transcribe the inserted intron RNA. The resulting cDNA copy of the intron is incorporated into the recipient DNA primarily by recombination mechanisms in yeast mitochondria (7) and by repair mechanisms in bacteria (8). Homing frequencies approach 100% for both fungal mitochondrial and bacterial introns (7, 8).

To initiate mobility, the intron-encoded RNP complex uses both its RNA and protein components to recognize specific sequences in its DNA target site (9-11). For the well-studied Lactococcus lactis L1.LtrB intron, the DNA target site extends from position -26 in the 5' exon (E1) to position +9 in the 3' exon (E2); positions numbered from the intron insertion site (Fig. 1A) (11):

Fig. 1A: Natural L1.LtrB DNA target sequence from -30 to +15 and base-pairing interactions with the intron RNA. Sequence elements IBS2 and IBS1 in the 5' exon and d' in the 3' exon of the DNA target are recognized primarily by base pairing with elements EBS2, EBS1, and d located in domain 1 of the intron RNA. The intron insertion site in the top (sense) strand and the endonuclease cleavage site in the bottom (antisense) strand are indicated by arrows.


A 14-nt region of the DNA target site (E1 -13 to E2 +1) is recognized primarily by base pairing with the intron RNA. This region includes short sequence elements denoted IBS2, IBS1, and d', which are complementary to intron sequences EBS2, EBS1, and d(IBS and EBS refer to intron and exon binding sites, respectively) (Fig. 1A, and B):

Fig. 1B. Schematic of the L1.LtrB intron showing base-pairing interactions EBS1-IBS1, EBS2-IBS2, and d-d' between the intron and flanking exons. The inset shows the location of the LtrA ORF and the T7 promoter introduced into intron domain IV in donor plasmids.

These same sequence elements are involved in base-pairing interactions required for RNA splicing (1). The regions of the DNA target site flanking the IBS and d' sequences are recognized by the IEP. The protein first recognizes a small number of nucleotide residues in the distal 5' exon region (E1 -26 to -11) and appears to cause local DNA unwinding, enabling the intron to form base pairs with the IBS and d' sequences for reverse splicing. Antisense-strand cleavage occurs after reverse splicing and requires additional interactions between the protein and 3' exon. The finding that at least a 14-nt region of the DNA target site is recognized by base-pairing with the intron RNA raises the possibility that group II introns can be retargeted to recognize any 14-nt DNA sequence, juxtposed to the fixed positions recognized by the IEP. By using crude target site recognition rules deduced from biochemical experiments, the L1.LtrB intron could in fact be retargeted to specific sites in a plasmid-borne E.colithyA gene. At best, however, these retargeted introns were very inefficient, presumably reflecting the cumulative effect of multiple changes from the norml target site sequence and/or additional constraints that must be satisfied for optimal base-pairing interactions (11).

To determine more detailed target site recognition rules, it is necessary to test a large number of different nucleotide combinations. For this purpose, we developed a new E. coli genetic assay (Fig. 1C) in which a modified L1.LtrB intron containing a phage T7 promoter near its 3' end is expressed from a T7lac promoter in a chloramphenicol-resistant (CamR) donor plasmid (pACD-LtrB) (12).

Fig. 1C. Genetic assay. The donor plasmid pACD-LtrB is a CamR pACYC184 derivative containing the full-length L1.LtrB intron and flanking exons, with a phage T7 promoter inserted downstream of the LtrA ORF in intron domain IV (12). The intron and flanking exon sequences (E1 and E2) are cloned behind a a T7lac promoter, and E. coli rrnB T1 and T2 transcription terminators are positioned downstream of the intron. The recipient pUCR-LtrB/Tet is a compatible AmpR  plasmid with an L1.LtrB target sequence (ligated ltrB exons E1 and E2) cloned upstream of a promoterless tetR gene (13). An E. coli rrnB T1 transcription terminator, which terminates both E. coli and T7 RNA polymerase, is inserted upstream of the target site, and an rrnB T2 terminator, which terminates E. coli but not T7 RNA polymerase, is inserted between the target site and the tetRgene. A phage T7 Tf terminator is inserted downstream of the tetR gene to terminate T7 RNA polymerase. Movement of the intron carrying the phage T7 promoter into the DNA target site activates expression of the tetRgene.


References and Notes:

1. Michel F, and Ferat JL, Annu. Rev. Biochem. 64: 435 (1995).

2. Lambowitz AM, Caprara MG, Zimmerly S, and Perlman PS, in "The RNA World", Gesteland RF, Cech TR, and Atkins JF, (Eds.), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, Ed. 2, 1999, pp. 451-485.

3. Zimmerly S, Guo H, Perlman PS, and Lambowitz AM, Cell 82: 545 (1995).

4. Zimmerly S, et al, Cell 83: 529 (1995).

5. Matsuura M, et al, Genes Dev. 11: 2910 (1997).

6. Yang J, Zimmerly S, Perlman PS, and Lambowitz AM, Nature 381: 332 (1996).

7. Eskes R, Yang J, Lambowitz AM, and Perlman PS, Cell 88: 865 (1997).

8. Cousineau B, et al, Cell 94: 451 (1998).

9. Guo H, Zimmerly S, Perlman PS, and Lambowitz AM, EMBO J. 16: 6835 (1997).

10. Yang J, Mohr G, Perlman PS, and Lambowitz AM, J. Mol. Biol. 282: 505 (1998).

11. Mohr G, Smith D, Belfort M, and Lambowitz AM, Genes Dev. 14: 559 (2000).

12. ...


Additional Reference:

"Oncogenes as Molecular Targets within Active Chromatin". 


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