Homing endonucleases: from basics to therapeutic applications

Abstract

Homing endonucleases (HE) are double-stranded DNAses that target large recognition sites (12-40 bp). HE-encoding sequences are usually embedded in either introns or inteins. Their recognition sites are extremely rare, with none or only a few of these sites present in a mammalian-sized genome. However, these enzymes, unlike standard restriction endonucleases, tolerate some sequence degeneracy within their recognition sequence. Several members of this enzyme family have been used as templates to engineer tools to cleave DNA sequences that differ from their original wild-type targets. These custom HEs can be used to stimulate double-strand break homologous recombination in cells, to induce the repair of defective genes with very low toxicity levels. The use of tailored HEs opens up new possibilities for gene therapy in patients with monogenic diseases that can be treated ex vivo. This review provides an overview of recent advances in this field.

Fig. 1

Homing mechanisms for group I introns (a), inteins (b) and group II introns (c). The homing endonuclease coding sequence of gene A (green) is duplicated in its cognate allele, gene A′. Mobile ORFs and encoded products are green; host gene exons and products are yellow; the other nucleic acid sequence is black, DNA is coloured with a 3D effect and RNA is flat. The ORF position is indicated with a black arrow. HE Homing endonuclease, M maturase, RT reverse-transcriptase, RNP ribonucleoprotein. The different stages of each mechanism are numbered in the scheme. The diagram showing the mechanism of action for group II introns is highly simplified. More detailed figures of their homing mechanisms can be found in specific reviews [158]

Fig. 2

Crystallographic structures of representative members of the five HE families. a LAGLIDADG family: monomeric I-DmoI and homodimeric I-CreI. b PD-D/E-XK family: the tetrameric I-SspI. c His-Cys Box family: the homodimeric I-PpoI. d GIY-YIG family: I-TevI catalytic and DNA binding domains. e HNH family: the monomeric I-HmuI. f Engineered heterodimeric variants Amel and Ini based on the I-CreI template. a–e The enzymes are shown in cartoon representation with the bound target site in stick representation. Catalytic ions are shown as yellow spheres and structural Zn ions are shown as orange spheres

Fig. 3

Double-strand breaks (DSBs) can be repaired by several homologous recombination (HR)-mediated pathways, including double-strand break repair (DSBR) and synthesis-dependent strand annealing (SDSA). Upper In both pathways, repair is initiated by resection of a DSB to provide 3′ single-stranded DNA (ssDNA) overhangs. Strand invasion at these 3′ ssDNA overhangs into a homologous sequence is followed by DNA synthesis at the invading end. Lower left After strand invasion and synthesis, the second DSB end can be captured to form an intermediate with two Holliday junctions (HJs). After gap-repair DNA synthesis and ligation, the resolved structure at the HJs may be a non-crossover (black arrowheads at both HJs) or crossover product (green and black arrowheads). Lower right Alternatively, the reaction may proceed to SDSA by strand displacement and the annealing of the extended single-strand end to the ssDNA at the other break end, followed by gap-filling DNA synthesis and ligation. The repair product from SDSA is always non-crossover

Fig. 4

Diagram showing the ex vivo approach. The location and characterization of chromosomal damage is followed by the introduction of the engineered HE in the isolated cells population together with the correcting template DNA matrix. The mutated gene, upon DBS-induced HR, is repaired and, after selection, cells with a normal gene are recovered

Fig. 5

Strategy for the making of redesigned HEs. a General strategy. I-CreI variant libraries with locally altered specificity are generated. These mutants were assembled into homodimeric and heterodimeric proteins using a combinatorial approach, generating meganucleases with fully redesigned specificity. b The RAG1 series of targets. Two intermediary palindromic targets were derived from the non-palindromic human gene target. These were used to select for the homodimeric I-CreI variants that served as scaffold for the specific heterodimer. The two 3 bp segments used in the library screening are boxed

Fig. 6

Targeted genes by modified endonucleases. a Human XPC (left panel) and RAG1 (right panel) genes showing the sequence recognized and cleaved by I-CreI amel [38] and I-CreI v2-v3 [27] variants which differ in 15 bp and 16 bp respectively from the original I-CreI 22 bp target (black box) [31]. The gene information was retrieved from http://www.ncbi.nlm.nih.gov/. b Tobacco acetolactate synthase gene showing the ZFN DSB site and the maximum endogenous repaired distance obtained by HR at a frequency of 2% [145]. c Human RAG1 gene showing the I-CreI v2-v3 DSB site and the donor DNA construct used to verify the 6% frequency of exogenous DNA integration, as a marker, in the genomic locus [27]. Red stars show clusters of mutations in the active core of the RAG1 protein that cause T-B-severe combined immune deficiency or Omenn syndrome [159]