Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
, 34 (22), e149

A Combinatorial Approach to Create Artificial Homing Endonucleases Cleaving Chosen Sequences

Affiliations

A Combinatorial Approach to Create Artificial Homing Endonucleases Cleaving Chosen Sequences

Julianne Smith et al. Nucleic Acids Res.

Abstract

Meganucleases, or homing endonucleases (HEs) are sequence-specific endonucleases with large (>14 bp) cleavage sites that can be used to induce efficient homologous gene targeting in cultured cells and plants. These findings have opened novel perspectives for genome engineering in a wide range of fields, including gene therapy. However, the number of identified HEs does not match the diversity of genomic sequences, and the probability of finding a homing site in a chosen gene is extremely low. Therefore, the design of artificial endonucleases with chosen specificities is under intense investigation. In this report, we describe the first artificial HEs whose specificity has been entirely redesigned to cleave a naturally occurring sequence. First, hundreds of novel endonucleases with locally altered substrate specificity were derived from I-CreI, a Chlamydomonas reinhardti protein belonging to the LAGLIDADG family of HEs. Second, distinct DNA-binding subdomains were identified within the protein. Third, we used these findings to assemble four sets of mutations into heterodimeric endonucleases cleaving a model target or a sequence from the human RAG1 gene. These results demonstrate that the plasticity of LAGLIDADG endonucleases allows extensive engineering, and provide a general method to create novel endonucleases with tailored specificities.

Figures

Figure 1
Figure 1
Design of the libraries of I-CreI variants: rationale. (a) Structure of I-CreI bound to its DNA target, according to Chevalier et al. (29), and localization of the area of the binding interface chosen for randomization in this study (green). The binding interface mutated in a former report, and including residues Q44, R68 and R70 is also represented (red). In the combinatorial approach described below, we combined the regions represented in green and red. (b) Zoom showing residues 28, 30, 33, 38 and 40 chosen for randomization. (c) Summary of I-CreI–DNA interaction in the external region of the I-CreI DNA target (in green on Figure 1a). The target represented, C1221, is a palindromic target cleaved by I-CreI (29). Only base specific contacts are indicated. The 10NNN (±8, ±9, ±10 nt, in green on Figure 1a) and 5NNN (±3, ±4, ±5 nt, in red in Figure 1a) regions of the target are boxed.
Figure 2
Figure 2
Identification of novel I-CreI derivatives with locally altered specificity. (a) Yeast screening assay principle. A strain expressing the meganuclease (MEGA) to be assayed is mated with a strain harboring a reporter plasmid containing the chosen target. The target is flanked by overlapping truncated LacZ genes (LAC and ACZ). Upon target cleavage, tandem repeat recombination restores a functional LacZ gene, which can be monitored by standard methods. (b) Examples of profiling. Each novel endonuclease is profiled in yeast on a series of 64 palindromic targets, differing from the sequence shown in Figure 1c at positions ±8, ±9 and ±10. These targets are arrayed as in Figure 2c. As described previously (11), blue staining indicates cleavage. (c) Numbers of mutants cleaving each target, and average intensity of cleavage. Each sequence is named after the −10, −9, −8 triplet (10NNN). The number of proteins cleaving each target is shown below, and the level of gray coloration is proportional to the average signal intensity obtained with these cutters in yeast.
Figure 3
Figure 3
Strategy for the making of redesigned HEs. (a) General strategy. A large collection of I-CreI derivatives with locally altered specificity is generated. Then, a combinatorial approach is used to assemble these mutants into homodimeric proteins, and then into heterodimers, resulting in a meganucleases with fully redesigned specificity. (b) Making of combinatorial mutants cleaving the COMB1 target: a workflow. Two palindromic targets are derived from the COMB1 targets, and homodimeric combinatorial mutants are designed to cleave these two targets. Positives are then coexpressed to cleave the COMB1 target. (c) The RAG1 series of target. Two palindromic targets are derived from RAG1.1. Then, a worflow similar to that described for the COMB series of target can be applied.
Figure 4
Figure 4
Secondary screening of combinatorial mutants cleaving COMB2. Upper panel: map of the mutants feature on the following panels. As described in text, combinatorial mutants are named with a eight letter code, after residues at positions 28, 30, 33, 38, 40, 44, 68 and 70 and parental controls with a five letter or three letter code, after residues at positions 28, 30, 33, 38 and 40 or 44, 68 and 70. Mutants are screened in yeast against COMB2 and 10TGC and 5GAC, the two parental targets.
Figure 5
Figure 5
Biochemical and biophysical characterization of combinatorial mutants. (a) Examples of raw data for in vitro cleavage (see Materials and Methods). Different concentrations of proteins were assayed. Lanes 1 to 15: protein concentrations in nM are 250, 189.4, 126.3, 84.2, 63.2, 42.1, 21.1, 15.8, 10.5, 7.4, 4.2, 2.1, 1.0, 0.5 and 0. (b) Cleavage of COMB2 by combinatorial mutants. (c) Cleavage of COMB3 by combinatorial mutants. (d) Thermal denaturation of the same proteins measured by CD. The bold line corresponds to I-CreI N75, with a mid point denaturation temperature of 65°C. Other proteins: KNHQS/KEG (mid point denaturation temperature: 65.3°C), KNHQS/KAS (64.9°C), KEG (63.1°C),KNHQS (62.2°C), NNSRQ (61.2°C), KAS (61.2°C), KAS (61.2°C), ARR (57.3°C), ASR (57.1°C), NNSRK/ARR (55.8°C), NNSRK/ASR (55.8°C). For protein nomenclature, see Figure 4.
Figure 6
Figure 6
Cleavage of non-palindromic target by redesigned heterodimers. (a) Cleavage of COMB1 by heterodimers (lower right panel). Cleavage of COMB2 and COMB3 palindromic targets by the parent homodimers is indicated on the top and left panel. For combinatorial mutants, nomenclature is the same as for Figure 4 and in text. (b) Cleavage of RAG1.1 target by heterodimers. As described in text, combinatorial mutants are named after 10 residues instead of 8, corresponding to positions 28, 30, 33, 38, 40, 44, 68, 70, 75 and 77.

Similar articles

See all similar articles

Cited by 103 PubMed Central articles

See all "Cited by" articles

References

    1. Thierry A., Dujon B. Nested chromosomal fragmentation in yeast using the meganuclease I-Sce I: a new method for physical mapping of eukaryotic genomes. Nucleic Acids Res. 1992;20:5625–5631. - PMC - PubMed
    1. Choulika A., Perrin A., Dujon B., Nicolas J.F. Induction of homologous recombination in mammalian chromosomes by using the I-SceI system of Saccharomyces cerevisiae. Mol. Cell. Biol. 1995;15:1968–1973. - PMC - PubMed
    1. Rouet P., Smih F., Jasin M. Introduction of double-strand breaks into the genome of mouse cells by expression of a rare-cutting endonuclease. Mol. Cell. Biol. 1994;14:8096–8106. - PMC - PubMed
    1. Puchta H., Dujon B., Hohn B. Two different but related mechanisms are used in plants for the repair of genomic double-strand breaks by homologous recombination. Proc. Natl Acad. Sci. USA. 1996;93:5055–5060. - PMC - PubMed
    1. Donoho G., Jasin M., Berg P. Analysis of gene targeting and intrachromosomal homologous recombination stimulated by genomic double-strand breaks in mouse embryonic stem cells. Mol. Cell. Biol. 1998;18:4070–4078. - PMC - PubMed

Publication types

Feedback