Skip to main content

Homing endonucleases from mobile group I introns: discovery to genome engineering

Abstract

Homing endonucleases are highly specific DNA cleaving enzymes that are encoded within genomes of all forms of microbial life including phage and eukaryotic organelles. These proteins drive the mobility and persistence of their own reading frames. The genes that encode homing endonucleases are often embedded within self-splicing elements such as group I introns, group II introns and inteins. This combination of molecular functions is mutually advantageous: the endonuclease activity allows surrounding introns and inteins to act as invasive DNA elements, while the splicing activity allows the endonuclease gene to invade a coding sequence without disrupting its product. Crystallographic analyses of representatives from all known homing endonuclease families have illustrated both their mechanisms of action and their evolutionary relationships to a wide range of host proteins. Several homing endonucleases have been completely redesigned and used for a variety of genome engineering applications. Recent efforts to augment homing endonucleases with auxiliary DNA recognition elements and/or nucleic acid processing factors has further accelerated their use for applications that demand exceptionally high specificity and activity.

Introduction

Homing endonucleases, also termed ‘meganucleases’, are highly specific DNA cleaving enzymes, found within all forms of microbial life as well as in eukaryotic mitochondria and chloroplasts, that are encoded by genes that display genetic mobility and persistence. The activity of these proteins is directly responsible for the genetic behavior of their corresponding reading frames, by inducing homology-driven gene conversion events at the site of the DNA double-strand break that result in invasion by the endonuclease gene. When the homing endonuclease gene is embedded within a self-splicing element (a microbial intron or intein), the homing endonuclease gene is further enabled with the ability to invade coding sequences within their hosts’ genomes. Studies of the genetic behavior of homing endonuclease genes and of the structure and function of their endonuclease gene products over the past several decades have provided enormous detail on their evolution and function, and have allowed several types of homing endonucleases to be engineered and used for applications that require targeted gene modification.

The discovery of mobile introns and their homing endonucleases dates back to the 1970s. In 1978, an intervening sequence within a yeast mitochondrial ribosomal DNA (rDNA) was visualized using electron microscopy [1]. A subsequent study [2] described the sequence and organization of this yeast element, concluding that the rDNA was interrupted by an insertion of approximately 1 kb. Taken together, these papers provided the initial details corresponding to a locus in the yeast mitochondrial genome, termed ‘omega’, that had previously been observed to display dominant, non-Mendelian inheritance in mating experiments, a phenomena that eventually became known as ‘homing’ (Figure 1) [3].

Figure 1
figure 1

Generalized homing mechanisms for mobile group I introns and inteins. In both cases, the activity of the endonuclease (which is translated either as a free-standing protein from the intron, or as a fusion with the surrounding intein) leads to a double-strand break in an allele of the host gene that does not contain the intervening sequence. Subsequent repair via homology-driven strand invasion and recombination and DNA replication, using the allele containing the intron or intein (as well as the associated endonuclease coding sequence), completes the homing process. HEG, homing endonuclease gene.

Within 2 years, the complete nucleotide sequence of that mobile element, corresponding to a group I intron, was determined from several yeast strains. These analyses indicated that the intron was exceptionally long (1,143 base pairs), and contained an apparent reading frame that might encode a 235 residue protein [4]. A comparison of rDNAs from divergent organisms demonstrated that introns with similar organizations appeared to exist at a variety of positions within that otherwise highly conserved host gene, leading to a suggestion that these elements were recent additions to their mitochondrial genomes.

A similar study of intron sequences in the yeast mitochondrial cytochrome b (cob) gene, reported in the same year, demonstrated the presence of a different intron-encoded protein that appeared to be responsible for “mRNA maturase” activity (a function required for splicing and maturation of the cytochrome b message) [5]. Thus, the presence and sequence of several intron-encoded protein factors, and hypotheses describing two different biological functions (intron homing and intron splicing) were established in the literature within a period of several months.

Shortly after the initial descriptions of these intron-encoded reading frames, studies of a seemingly unrelated biological phenomenon provided the first hints of a biochemical mechanism that would eventually be linked to the process of intron homing [6, 7]. Termed the ‘yeast mating type switch’, this process required the action of a site-specific endonuclease (at the time, termed ‘YZ endonuclease’) that was found to drive the homology-driven conversion of the yeast mating type (MAT) locus. A gene at that site encodes a transcription factor which activates either of two different suites of genes that control MAT: the DNA cleavage event driven by this endonuclease-induced recombination between MAT and a ‘hidden MAT’ locus. In subsequent years, the YZ endonuclease was renamed the HO endonuclease, and found to belong to the LAGLIDADG protein family. The observations in these early studies, which pre-dated the first biochemical characterization of a LAGLIDADG homing endonuclease, reported many of the eventual hallmarks of their properties, including the description of a long DNA target site and the observation of variable fidelity at several base-pair positions within that site. The actual notation of the conserved LAGLIDADG protein motif, which is found in many proteins involved in intron mobility, intron splicing and MAT gene conversion, was subsequently facilitated by the identification and sequencing of a sufficient number of intron-encoded proteins to allow its identification [8].

In 1985, several research groups demonstrated that translation of an intron-encoded protein, from the large rDNA gene in Saccharomyces cerevisiae mitochondria, was required and responsible for that intron’s mobility and inheritance, and that a double-strand break formed in vivo either at or near the site of the intron insertion was required for that process [911]. While these investigators noted that this intron behavior was somewhat similar to transposase function, they further indicated that the homing process appeared to correspond to a non-reciprocal recombination event at the cleavage and intron insertion site (that is, at ‘omega’), and was accompanied by co-conversion of DNA sequence tracts flanking the intron. Subsequently, the catalytic activity and specificity of the protein, and its probable role in creating a double-strand break at omega, was demonstrated using purified recombinant protein [12]. Subsequent analyses firmly established that the protein appeared to recognize a target site spanning approximately 20 base pairs in length, and demonstrated that the protein displayed a complex pattern of recognition fidelity across that target site [13].

While many of the seminal observations regarding homing endonuclease function were made using genetic information and systems derived from fungal mitochondria, additional studies on similar mobile elements in algal chloroplasts further demonstrated their ubiquitous distribution and the generality of their ability to invade host genes. In particular, studies of the I-CreI homing endonuclease from Chlamydomonas reinhardtii further established the roles played by the enzyme and a surrounding intron in genetic mobility and persistence, as well as reinforcing the concept that flanking homology regions near the site of an enzyme-induced double-strand break are critical for gene conversion [1416]. Subsequent analyses of the distribution inheritance of additional mobile introns and homing endonucleases derived from algal chloroplasts, such as I-CeuI and I-MsoI, demonstrated that organellar genomes (and in particular, their rDNA genes) are often densely populated with such mobile elements [1719].

In 1990, an examination of an unusual gene structure encoding a yeast vacuolar ATPase led to the discovery of a novel form of splicing, in which the intervening sequence was translated in-frame with the surrounding host gene, and then precisely excised (without the aid of auxiliary factors) post-translationally [20]. Found within that element, which was eventually termed an ‘intein’, was a sequence that again harbored sequence similarity to the LAGLIDADG protein family. Similar to the role of intron-encoded endonucleases in homing, the resulting protein product (an in-frame fusion of endonuclease and surrounding intein) was found to be responsible for the mobility and invasiveness of the entire intervening sequence [21]. The corresponding protein construct (eventually named ‘PI-SceI’) eventually served as one of the first homing endonucleases to be characterized mechanistically [22, 23].

While much of the basic molecular biology of mobile introns and their homing was established by studies of intervening sequences isolated from organellar genomes in fungal and algal hosts, a series of subsequent studies using phage-derived mobile introns were critical for firmly establishing several additional details of that process. Shortly after the discovery of introns within phage genomes [24], investigators determined that many of them display mobility that is the result of intron-encoded homing endonucleases [25]. The ability to conduct quantitative homing assays using phage, both as a gene delivery vehicle and as a genetic recipient for mobile introns, allowed investigators to systematically characterize the mechanism and efficiency of intron transfer events to recipient alleles. These experiments demonstrated: (i) that homing is associated with co-conversion of flanking sequences that reflect the recombination process involved in the process of intron mobility [26]; and (ii) that the homing event does not specifically require the actual presence of an intron or intein, but instead is dependent only upon the expression of the endonuclease, the presence of its target site in acceptor DNA, the presence of sufficient homology between the DNA acceptor and donor, and the availability of phage- or host-encoded recombinase and exonuclease activities [27]. Subsequent studies demonstrated that intron mobility occurs in the context of phage recombination-dependent replication, and that homology-driven intron transfer can occur via multiple competing strand invasion pathways [28].

Review

Structures, functions and mechanisms

The experiments described above provided the initial examples of mobile group I introns and their corresponding homing endonucleases. Subsequent studies extending through the late 1990s demonstrated that similar mobile elements, each driven by intron-encoded proteins, are encoded across a vast array of organellar genomes, microbial genomes (including eubacteria, archaea, fungi, algae, and protists), and phage (see [29] for a review written during that time, and [30] for an additional review written this year). The transfer, duplication and transmission of these sequences was shown to be extremely efficient, leading to unidirectional gene conversion events in diploid genomes [9], possible horizontal transfer between phage and eukarya [31, 32], competition between mobile introns in mixed phage infections [33], movement of introns between different subcellular compartments in unrelated organisms [34] and the rapid spread of mobile introns into related target sites throughout a broad range of biological hosts [35]. Although homing endonucleases can also be encoded by free-standing reading frames, their association with self-splicing sequences frees them to invade highly conserved sequences in protein- and RNA-encoding host genes, and then to persist in microbial genomes that are otherwise subject to selective pressure to eliminate extraneous genetic elements [36]. The sheer number and density of homing endonucleases and associated introns found to occupy various genomes and host genes can be extremely high. For example, the genome of T4 phage is found to contain 12 free-standing and 3 intron-encoded homing endonuclease genes (encompassing 11% of the total coding sequence in that phage’s genome) (reviewed in [37]).

At least six unique families of group I homing endonucleases (‘LAGLIDADG’, ‘HNH’, ‘His-Cys box’, ‘GIY-YIG’, ‘PD-(D/E)xK’ and ‘EDxHD’ proteins) have been described over the past 25 years (reviewed in [38]). Each is named based on the presence of conserved sequence motifs that correspond to conserved structural and catalytic residues in each family’s catalytic domain and active site, and each is largely (although not absolutely) confined to a well-defined host range. Structural analyses of members from each of these families (Figure 2) demonstrate that they have embedded their nuclease catalytic cores in a wide variety of surrounding protein scaffolds, and appear to be descended from multiple, unique ancestral nucleases.

Figure 2
figure 2

Representative structures of homing endonuclease families and subfamilies. Top: three separate types of catalytic nuclease domains (GIY-YIG, HNH and EDxHD) are found in various phage-encoded homing endonucleases (as well as less commonly in organellar genomes). As illustrated by the structure of full-length HNH endonuclease I-HmuI (middle), these nuclease domains are coupled to elongated DNA-binding regions that are involved in contacts to long target sites. Shown in the insets are crystal structures of the catalytic domains of the GIY-YIG endonuclease I-TevI (left) and the EDxHD endonuclease I-Bth0305I (right). Both of those endonucleases display a general domain organization that is similar to that of I-HmuI: a nuclease cleavage domain tethered to an extended DNA binding region that contains multiple structural motifs. Middle: two closely related types of LAGLIDADG homing endonucleases, corresponding to homodimeric and monomeric enzymes, are encoded within organellar and archaeal genomes. Whereas the homodimeric enzymes can be applied to genome engineering after converting their quaternary organization into an artificial monomeric protein (by tethering the two equivalent domains to one another with a peptide linker) the wild-type monomeric enzymes can be used directly for that purpose. In either case, the N- and C-terminal domains of the protein can be individual engineered and then fused to create highly specific gene targeting proteins. Bottom: His-Cys box endonucleases (which harbor a variant of the HNH active site) and PD-(D/E)xK endonucleases are found in protist and cyanobacterial genomes. Both enzymes are multimers (a homodimer and a tetramer, respectively).

A hallmark of all homing endonucleases, regardless of their family origin, is the contrast between their small size (homing endonuclease genes usually encode proteins that comprise fewer than 200 residues) and their long DNA target sites (which often extend to well over 20 base pairs). The determination of the first structures of representatives from each of these homing endonuclease families [3950] illustrated two strategies that allow such compact proteins to bind long DNA sequences. The majority of homing endonucleases that are most commonly derived from phage (that contain either an HNH [46], a GIY-YIG [49] or an ‘EDxHD’ [47] catalytic domain) form highly elongated proteins with minimal hydrophobic cores. They rely upon the presence of additional DNA binding regions (often termed Nuclease-associated modular DNA-binding domains or ‘NUMODs’ [51]) that are loosely tethered to their catalytic domains, and thereby contact DNA target sites up to 30 base pairs in length. At least one of these phage-derived homing endonuclease families (the most recently described ‘EDxHD’ enzymes, exemplified by the I-Bth0305I endonuclease) appears to employ both of these strategies, by forming a long multi-domain structure while also dimerizing on an exceptionally long DNA target that extends to nearly 60 base pairs in length [47].

In contrast, many homing endonucleases found in archaea, eukarya and eubacteria (corresponding to the LADLIDADG [43], His-Cys box [41] and PD-(D/E)xK [50] proteins) display more compact protein folds that usually multimerize and thereby double their DNA-contact surface. This strategy constrains the endonuclease to recognition of a DNA sequence that contains significant palindromic symmetry. Only one subfamily of homing endonucleases (the monomeric LAGLIDADG enzymes) display compact, globular protein folds and also recognize completely asymmetric DNA target sites [39, 45]. Those enzymes are among the largest of homing endonucleases, often containing nearly 300 residues that are distributed across two pseudo-symmetric protein domains.

DNA recognition

Analyses of insertion sites for mobile group I introns and the corresponding cleavage sites for their homing endonucleases indicate that they are often found at positions and sequences within their host genes corresponding to coding sequences that span critical residues within an enzyme active site, a ligand-binding pocket, or a strongly conserved structural motif. In one particularly notable example, an exceptionally well-conserved sequence in a large rDNA, that encodes a structural helix at the ribosomal RNA interface and is located near a transfer RNA (‘tRNA’) binding site and the peptidyl transferase active site, has been independently invaded three times: in protists by introns armed with His-Cys box homing endonuclease genes [52], in archaea by introns armed with LAGLIDADG homing endonuclease genes [53] and in metazoans by a retrotransposon [54]. Thus, a sequence that is sufficiently invariant over the course of evolution can become a repeated target for invasion by mobile elements, including homing endonucleases.

DNA recognition mechanisms vary widely across the families of homing endonucleases described above, but in each case these mechanisms strike a balance between the somewhat orthogonal requirements of (i) recognizing a target of sufficient length to avoid overt toxicity in the host, while (ii) accommodating at least a small amount of sequence drift within that target. The LAGLIDADG and His-Cys box enzymes, which are the most sequence-specific of these proteins, rely upon extensive, antiparallel DNA-binding β-sheets that dock into the major grooves of their target sites [41, 43, 55]. Nearly one-quarter of the amino acids in the entire endonuclease participate in the resulting protein-DNA contacts. There they establish a collection of sequence-specific and non-specific contacts that comprise many directional hydrogen bonds to individual bases, water-mediated contacts, and additional steric contacts that further enforce specificity. These contacts are distributed non-uniformly across base pairs throughout the target site. DNA bending near the middle of each target appears to further contribute to sequence-specificity.

In contrast, the less specific homing endonucleases, found primarily in phage, often form a more heterogeneous collection of DNA contacts within the major and minor groove, as well as across the backbone, of their target sites. These enzymes (typified by I-TevI (a GIY-YIG endonuclease) [49], I-HmuI (an HNH endonuclease) [46] and I-Bth0305I (an ED-HD endonuclease) [47]) all display extended, multi-domain protein structures in which disparate structural elements that include individual α-helices, zinc fingers and/or helix-turn-helix domains. These regions of the proteins contact DNA targets that can span up to 30 base pairs. Although the overall specificity of these endonucleases is considerably lower than their eukaryotic and archaeal cousins, these endonucleases also can display elevated specificity at base pair positions within the target that are strongly constrained in the host gene [47, 5658].

The specificity profiles and overall frequency of DNA cleavage exhibited by homing endonucleases has been particularly well characterized for the LAGLIDADG family, which comprise the most specific of the homing endonucleases and are most commonly used for applications in gene targeting and genome engineering. Studies of the target sites and specificities of three of the earliest identified examples of these enzymes (the monomeric I-SceI endonuclease, the homodimeric I-CreI, and the intein-encoded PI-SceI) each indicated that the overall length of their target sites was 20 base pairs or greater. Their fidelity of recognition, as evaluated by the effect of base-pair substitutions within the target on cleavage activity, was highly variable across the target site [5961]. Subsequent comparison of the I-CreI specificity profile with the distribution of atomic contacts throughout the protein-DNA interface indicated that specificity was largely derived by a large number of direct and water-mediated contacts between protein side chains and nucleotide bases, particularly across a series of at least 3 consecutive base pairs in each half-site [55]. In addition, the bending and distortion of the DNA target near the center of the site appears to elevate total target specificity and contribute heavily to cleavage fidelity at the four ‘central’ base pairs that reside between the scissile phosphates on each DNA strand (LAGLIDADG enzymes always generate a pair of 4-base, 3’ overhangs).

Subsequently, a series of much more detailed analyses of the specificity profile of a single LAGLIDADG enzyme (I-AniI) were conducted, utilizing three separate, complementary approaches: i) an in vitro selection experiment for cleavable substrates, extracted and amplified from a library of randomized target site variants [62]; ii) direct examination of relative binding affinity and cleavage activity for the enzyme against all single base pair variants of the enzyme’s target site using surface-display and flow-assisted cell sorting (FACS) analyses of metal-dependent binding and cleavage [63]; and iii) direct examination of relative binding affinity and cleavage activity against all single base pair variants of the enzyme’s target site, using purified enzyme and corresponding biochemical measurement of relative cleavage rates and binding affinities [64].

These experiments, in addition to thoroughly characterizing the specificity of one particular homing endonuclease, provided considerable insight into the behavior of LAGLIDADG enzymes. Collectively, the experiments indicated that:

  1. 1)

    The overall specificity of the enzyme, in terms of expected cleavage frequency versus random DNA target sequences, is approximately 1 in 108 (and possibly somewhat more specific, because the extent to which base pair substitutions, that are individually tolerated by the enzyme, would be accommodated simultaneously is unclear).

  2. 2)

    The positions within the target site where base pair substitutions are particularly well-tolerated by the enzyme, corresponding to ‘promiscuous’ recognition, are well-correlated with loosely constrained ‘wobble’ positions in the coding frame of the underlying host gene (the mitochondrial cytochrome B oxidase gene in Aspergillus nidulans).

  3. 3)

    Many substitutions in the target site that cause decreased cleavage activity often do so primarily via a reduction in substrate binding affinity or through a reduction in substrate cleavage rate. In the case of I-AniI, these two different effects map rather cleanly to the two DNA-half sites, and appear to reflect an inherent asymmetry in the role of each protein domain (and the corresponding DNA half-sites) in target site binding and cleavage.

Additional data on the in vivo specificity of homing endonucleases, and whether their activity profiles differ significantly from those measured using in vitro methods, are relatively scarce. However, at least one recent analysis of the apparent cleavage targets of I-SceI in transfected human cells [65] has indicated that, for at least one LAGLIDADG enzyme, a significant number of chromosomal target sites (including some that differ significantly from the canonical sce target sequence) appear to be cleaved.

DNA cleavage

Many of the mechanisms and corresponding active site architectures by which a phosphodiester bond can be hydrolyzed [66] are observed for the various families of homing endonucleases (Figure 3). For all of these enzymes, the reaction proceeds according to a metal-dependent hydrolysis reaction, without the formation or accumulation of a covalent enzyme-DNA intermediate. Biochemical and structural analyses indicate that they all utilize an activated water molecule as the incoming nucleophile, which drives an in-line SN2 displacement of the 3’ leaving group, resulting in the formation of 5’ phosphate and 3’ hydroxyl product ends. They utilize either a strong general base to deprotonate the incoming water molecule, and/or a bound metal ion to significantly decrease the pKa of the water molecule, as well as an appropriate electropositive group positioned to stabilize the phosphoanion transition state and a proton donor to neutralize the 3’ hydroxylate leaving group.

Figure 3
figure 3

Representative active sites and generic mechanisms of DNA cleavage by homing endonuclease families. The HNH and His/Cys box endonucleases contain similar nuclease motifs and active sites, and are thought to be related via divergence from a common ancestor. In those enzyme families, an absolutely conserved active site histidine residue directly deprotonates a water molecule; the ability of the histidine side chain to act as a general base is facilitated by a hydrogen bond to a neighboring carbonyl moiety (usually an asparagine side chain). The GIY-YIG endonucleases use a similar mechanism, with the difference that an active site tyrosine appears to serve a similar role as an activated general base, again to deprotonate the incoming nucleophilic water molecule. In contrast, the PD-(D/E)xK and EDxHD endonucleases display similar active site structural motifs and mechanisms that appear to be similar to previously well-characterized type II restriction endonucleases; in those enzymes a metal-bound water molecule acts as the incoming nucleophile. In these enzymes (corresponding to either the restriction or the homing endonuclease catalysts) the precise number of metal ions employed is often not entirely clear (and hence is represented in the figure either as a single-metal or a two-metal-dependent active site). In each panel of the figure, the most conserved catalytic elements (corresponding to those regions that contain the enzymes’ namesake motifs) are shown in red, and the corresponding secondary structural elements of the catalytic cores are labeled. LH1 and LH2 in the middle panel refer to LAGLIDADG helices 1 and 2 in a monomeric LAGLIDADG homing endonuclease.

Different homing endonuclease families display different strategies by which these basic mechanistic requirements of a nuclease reaction are satisfied (Figure 3). The LAGLIDADG, PD-(D/E)xK and EDxHD nucleases all appear to utilize variations of a traditional two-metal hydrolysis reaction, in which a metal-bound hydroxyl serves as a nucleophile, and a second bound metal ion is appropriately positioned to stabilize the phosphoanion and the 3’ leaving group [47, 67, 68]. The LAGLIDADG active site is particularly unique in that: (i) the nucleophilic, metal-bound water is surrounded solely by a network of additional ordered solvent molecules, rather than being directly coordinated by protein side chains; and (ii) the two active sites (which are closely juxtaposed in order to cleave phosphates on either side of the DNA minor groove) often appear to share a common metal ion [69].

In contrast, the HNH, His-Cys Box and GIY-YIG endonucleases all appear to follow a reaction mechanism in which the incoming water molecule is not associated with a bound metal ion, but instead is in direct contact either with the side chain of a conserved histidine or tyrosine residue [46, 48, 70]. In either case, the activation of the nucleophilic water molecule require a strong enough general base to abstract a proton from a water molecule that is not associated with nearby metal ion.

Additional and related functions

The most obvious biological function of a homing endonuclease is to drive the mobility, invasiveness, and persistence of its own coding sequence; as such the protein is the product of a specific form of ‘selfish DNA’. However, this function is largely independent of host-derived selection pressure, because the mere presence and persistence of a mobile intron does not provide any obvious benefit to the host. As a result, mobile introns and their associated homing endonuclease genes are observed to undergo a relatively rapid evolutionary cycle of invasion, mutational degradation of its form and function, and precise deletion from the host genome (which produces a site that is once again subject to invasion) [71].

Perhaps as a way to increase selection pressure for maintenance of a stable protein fold after gene invasion, some homing endonucleases have acquired an additional biological function that may provide a benefit to the host. The most well-documented of these functions, termed ‘maturase’ activity, corresponds to the direct interaction of the intron-encoded protein with the surrounding intron - a specific, high-affinity binding activity that is required to ‘chaperone’ the RNA element through required steps of folding that lead to its eventual splicing [5]. In some cases, closely related intron-encoded proteins may each display only a single activity (that of either an endonuclease or a maturase) [72]; in at least one case only a single amino acid substitution in a monofunctional maturase was needed to restore endonuclease activity [73]. In other cases, an intron-encoded protein may display bifunctionality, acting both as an active endonuclease and as a maturase. In the most well studied example of such a dual-function protein (the I-AniI endonuclease/maturase) the surfaces and residues of the protein involved in DNA and RNA recognition were found to be different, and the two activities could be uncoupled through separate point mutations that disrupted each activity [39, 74].

Another system in which a homing endonuclease displays a secondary function with potential benefit to the host is the T4 phage-encoded I-TevI endonuclease, which displays not only DNA cleavage activity, but also acts as a transcriptional autorepressor of its own expression [75]. This secondary activity depends upon the endonuclease binding a DNA sequence that overlaps a late promoter within the 5’ region of its own reading frame - a function that is essential for optimal splicing activity of the surrounding intron, in order to avoid compromising the expression of the host gene. The cis regulatory sequence that is recognized by the DNA binding domain of I-TevI is similar, but not identical, to comparable base-pair positions in the enzyme’s cleavage target. However, the absence of an appropriately positioned upstream consensus sequence (5’-CNNNG-3’) for recognition by the nuclease domain greatly reduces the cleavage activity of the enzyme at the regulatory site, allowing the I-TevI protein to function as a transcriptional repressor.

Beyond the acquisition of secondary functions and activities by active homing endonucleases, there are clear evolutionary connections between these proteins and a wide variety of host proteins and functions (recently reviewed in [76]). Proteins that share common folds and catalytic motifs with homing endonucleases are found in proteins that participate in phage restriction, in DNA repair, in processing DNA junctions and cross-over structures during strand invasion events that lead to integration, transposition and recombination, in transcriptional regulation, in chromatin remodeling and maintenance, and in gene conversion events. While the relative origin(s) and sequence of events that led to the establishment of modern day homing endonucleases and related host proteins is not obvious, it seems clear that a small number of ancient DNA binding nucleases have served as common ancestors for a wide variety of proteins that are engaged in competing functions of genomic invasion and genomic fidelity.

Application for genome engineering

Genome engineering and targeted gene modification is a rapidly maturing discipline in which genomes within cell lines, tissues or organisms are manipulated and altered at specified individual loci [77]. The first demonstrations that the introduction of a site-specific nuclease into a mammalian genome could increase the efficiency of a site-specific sequence conversion event were conducted using the I-SceI LAGLIDADG endonuclease [7880]. In those studies, the wild-type target site of I-SceI was first introduced into a desired chromosomal allele, prior to the subsequent introduction of the endonuclease. While this strategy did little to simplify the process of targeted gene modification, it demonstrated that highly specific endonucleases that generated double-strand breaks at unique loci in complex eukaryotic genomes could greatly enhance the efficiency of corresponding gene modification processes at those positions. Within 2 years of those studies, the first artificial zinc-finger nucleases (ZFNs) had been described [81], and the race to develop and apply them for specific genome editing purposes had begun.

Four separate macromolecular scaffolds, which each generate site-specific double-stranded DNA breaks, can now be used for targeted gene modification: ZFNs (first described as genome editing tools in [82, 83]); transcriptional activator like (TAL) effector nucleases (TALENs) [84]; the clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 (‘CRISPR’) system [8587]; and LAGLIDADG homing endonucleases (now also termed ‘Meganucleases’) [88]. Thus, the field of site-specific genome engineering using site-specific nucleases enjoys a wealth of molecular scaffolds. Three are protein based and one relies on RNA-guided specificity for gene targeting.

The ease of constructing CRISPR-based gene targeting nucleases (and, to almost the same extent, of constructing TAL nucleases) has led to an explosion of activity in the field of nuclease-induced targeted gene modification experiments, and corresponding excitement concerning the potential of targeted genome engineering [89]. In contrast, the utility of LAGLIDADG homing endonucleases has been somewhat dismissed, on the assumption that the ‘degree of difficulty’ for retargeting their recognition profiles for a desired genomic target is too high (because their DNA recognition mechanisms cannot be reduced a simple modular ‘code’) (Figure 4). For the construction of genetically altered (‘transgenic’) model organisms and corresponding cell lines for research, this outlook is mostly appropriate. However, for therapeutic applications, which demand the highest level of targeting specificity, combined with high levels of gene modification activity, the continued development of compact, highly specific nuclease domains as an alternative to nonspecific nuclease domains that rely upon additional DNA targeting moieties seems appropriate. A recent proof of principle has demonstrated the possibility of replacing the R.FokI nuclease domain with the catalytic domain of the I-TevI homing endonuclease for the purpose of creating a site-specific, single chain nuclease with elevated specificity at the actual point of DNA cleavage [90], as well as experiments that have fused the more specific R.PvuII nuclease domain to TAL effector or zinc finger DNA binding domains [91, 92].

Figure 4
figure 4

Redesign of a LAGLIDADG homing endonuclease (HE; also termed a ‘meganuclease’) for a specific genome engineering application (such as modification of a disease-associated human gene locus) involves the alteration of a substantial fraction of its DNA-contacting residues, as well as further optimization of neighboring positions on the protein scaffold.

Soon after the original ‘proof of concept’ studies with I-SceI [7880], it became obvious that modification of a homing endonuclease’s cleavage specificity would be required in order to target and modify endogenous target sites in various biological genomes. The determination of the first DNA-bound structures of homing endonucleases (I-PpoI and I-CreI in 1998 [43, 93] and then I-MsoI, I-AniI and I-SceI in 2003 [39, 45, 55]) allowed identification of the amino acids in each system that were found within contact distance of base pairs in their DNA targets, both individually and within distinct ‘clusters’. Armed with such information, a series of experiments of increasing complexity, all designed to alter the DNA cleavage specificity of homing endonucleases, were reported, eventually leading to the ability to completely retarget homing endonucleases for the modification of unique genomic targets.

(i) Alteration of homing endonuclease target specificity at individual base pairs

Early studies provided multiple examples where mutation of individual residues in a homing endonuclease DNA-binding surface resulted in a change in the specificity at a single position in the target site [60, 94]. The earliest experiments to alter LAGLIDADG endonuclease specificity often relied upon in vitro or cellular assays to visually identify mutated endonuclease constructs that displayed altered recognition specificity. Some of these early protocols utilized reporters of high affinity DNA binding (for example, through the use of a bacterial two-hybrid screening strategy) [60] or methods that coupled endonuclease activity to the elimination of a reporter gene [94, 95].

At the same time, an experiment that relied on structure-based redesign of the protein-DNA interface to alter specificity at a single base pair, relying upon computational algorithms that repack and optimize new protein-DNA contacts, was also reported [96]. In that study, the redesigned enzyme bound and cleaved a corresponding recognition site harboring a single base pair substitution 104 times more effectively than did the wild-type enzyme, with a level of target discrimination comparable to the original endonuclease.

(ii) Combined alteration of specificity at multiple, adjacent base pairs

By 2004, it was apparent that, in some cases, alteration of individual DNA-containing side chains in homing endonucleases might result in desirable and useful changes in specificity at individual base pairs in the target [97]. However, it was not clear to what extent such alterations might be combined in ‘additive’ steps for a complete redesign process, to more significantly alter the protein’s DNA binding and cleavage specificity. As a way forward, a particularly powerful selection method to screen a homing endonuclease library for altered DNA cleavage specificity was described in 2005, in which the protein’s cleavage activity was coupled to the homology-driven reconstitution of a reporter gene [98]. This method was used to systematically screen multiple semi-randomized libraries of the I-CreI homing endonuclease, where each library harbored collections of amino acid substitutions within ‘modules’ or ‘clusters’ of residues that collectively contacted several adjacent DNA base pairs. By doing so, investigators could isolate and characterize a large number of individual protein variants, harboring multiple amino acid changes that could accommodate multiple adjacent base pair substitutions at several distinct regions of the enzyme’s target site [99, 100].

Aside from building up a large collection of variants of the I-CreI enzyme that could cleave DNA target sites harboring many different clusters of altered base pair sequences, these studies also demonstrated that the output of such screens was more complex than might be predicted based on prior studies of changes to single amino acids in the protein-DNA interface. Alteration of individual protein side chains that caused reduced activity or specificity were sometimes well tolerated in more extensively altered pockets; conversely, some alterations of protein-DNA contacts that behaved well on their own were found to be incompatible with substitutions at adjacent positions (reviewed in [101]).

A separate effort, again conducted using structure-based computational redesign methods, to create a similar specificity change that involved multiple consecutive base pairs also recapitulated the results of the selection-based experiments [102]. The concerted redesign of the I-MsoI homing endonuclease to accommodate base pair substitutions at three consecutive positions was more successful than attempts to employ incremental or sequential redesign for recognition of individual substitutions, highlighting the importance of context-dependent optimization of protein-DNA interactions. Crystallographic structure analyses of all the redesigned enzymes in this study indicated that the basis of this behavior could be observed in patterns of structural context-dependence, extending across a local network of adjacent side chains and corresponding DNA base pairs, that caused unpredictable differences in DNA backbone conformation and side chains rotamers.

(iii) Domain shuffling

The concept that domain swaps between different wild-type homing endonucleases might be possible could greatly increase the number of such scaffolds for genome engineering (in theory, shuffling the N- and C-terminal domains of 10 wild-type endonucleases could yield up to 100 unique DNA-cleaving proteins with different target specificities). At the same time that the experiments described above were being performed, several studies demonstrated that entire domains or subunits from unrelated LAGLIDADG enzymes could be mixed and fused to create novel chimeric homing endonucleases that recognize corresponding chimeric DNA target sites [103105]. These studies demonstrated that the individual domains and subunits of LAGLIDADG enzymes are largely responsible for the recognition and binding of individual DNA half-sites. Subsequent experiments reinforced this conclusion [103, 106108]. Most importantly, these studies demonstrated that the task of altering a homing endonuclease’s cleavage specificity could be ‘broken down’ into two separate redesign projects to individually target the left and right half-sites of a DNA target, by systematically altering the DNA-contacting residues of the protein’s N- and C-terminal domains and then combining the final solutions for each domain into a single gene targeting protein.

To further explore this concept, more recent studies focused on systematic exchange of domains between homing endonucleases selected from a relatively closely related clade (all from mesophilic fungal mitochondrial genomes, with 40 to 70% sequence identity between the individual proteins) [109]. Using a simple method in which limited variation was introduced into the domain interface, catalytically active enzymes were recoverable for approximately three-fourths of the resulting chimeras. While potentially useful for future creation of large numbers of gene targeting reagents, such domain fusions will probably prove to be largely unnecessary for genome engineering, because several research groups have demonstrated that such reagents can now be readily obtained starting from wild-type proteins, as described in the next section.

(iv) Complete retargeting of homing endonucleases and application to genome editing

Multiple groups (both academic and within the biotech industry) have recently exploited the data summarized above to generate and use completely retargeted and optimized homing endonucleases for genome engineering and targeted gene modification. The method employed by these groups can loosely be divided into strategies that either ‘go deep’ (by focusing on the maximum possible ‘redesignability’ of a single homing endonuclease) or that ‘go broad’ (by employing bioinformatics to choose from an increasing number of well-characterized wild-type endonucleases, followed by the redesign of the most appropriate starting scaffold for a given target). Both approaches have been shown to work, and in both cases the use of direct structure-based redesign and structure-based selection methods have each found their place as part of the engineering process. A survey of the recent literature demonstrates the increasing pace and speed at which highly active and extremely specific gene-targeting reagents can be generated from LAGLIDADG homing endonucleases.

Two separate biotechology companies, Cellectis Inc. (8 rue de la Croix Jarry 75013 Paris, France) and Precision Biosciences Inc. (302 East Pettigrew Street Durham, NC 27701 USA)) have each described the creation of extensively altered variants of the I-CreI homing endonuclease and their successful application for nuclease-driven, targeted gene modification. Because wild-type I-CreI is a natural homodimeric enzyme, both efforts rely upon the ‘monomerization’ of the I-CreI protein to create a single-chain reagent in which the two subunits of the enzyme are linked by a peptide tether and then expressed in cis as a monomeric scaffold [110112]. Armed with this construct, redesign efforts can then be conducted on individual protein domains (targeting corresponding half-sites of the desired genomic target) with the resulting constructs combined into a single polypeptide which is further optimized for optimal in vivo performance. The strategies used to identify and combine individual amino-acid substitutions in the I-CreI scaffold differ between these two approaches. One group relies predominantly upon direct structure-based redesign of the wild-type protein [110], while the other relies upon the output of phenotypic screens from semi-randomized protein libraries [98]. Both approaches have largely converged on alteration of the same DNA-contacting protein side chains.

Using these approaches, these groups have created and employed redesigned variants of single-chain I-CreI endonuclease for a wide variety of purposes, such as modification and correction of the human XPC gene for the treatment of xeroderma pigmentosum [88, 113, 114], creation of cell lines harboring defined genetic insertions and alterations [115, 116], generation of transgenic lines of maize containing heritable disruptions of the ligueleless-1 and MS26 loci [110, 117], excision of defined genomic regions in Arabidopsis[118], insertion of multiple trait genes in cotton [119], generation of Rag1 gene knockouts in human cell lines [111, 120] and in transgenic rodents [121], disruption of integrated viral genomic targets in human cell lines [122], and demonstration of the correction of exon deletions in the human DMD gene associated with Duchenne Muscular Dystrophy [123].

Yet another biotechnology company (Pregenen Inc. (454 N. 34th St. Seattle WA 98103 USA)) has employed both a different homing endonuclease scaffold (I-OnuI, which is a naturally occurring monomeric LAGLIDADG enzyme, rather than a ‘monomerized’ homodimeric protein) and a considerably different in vitro engineering pipeline that relies upon yeast surface-display and high-throughput flow cytometry to screen semi-randomized endonuclease libraries for altered binding and cleavage specificity [63]. Using this strategy, gene targeting nucleases have been created that drive the disruption of fertility-related genes as part of a gene drive strategy for the control of insect disease vectors [124], and that quantitatively disrupt the T-cell receptor α-chain gene (as part of a broader strategy to create engineered T-cells that can be used as anticancer immunotherapeutic reagents) [125]. Unlike the engineering strategies employed for I-CreI, which both rely upon relatively low-throughput screening of enzyme variants and/or minimally complex libraries that are reliant upon prediction of specificity-changing amino acid substitutions at direct contact points between protein and DNA, the platform used with I-OnuI relies upon the elevated throughput that can be realized through the use of yeast (a naturally recombinant host that facilitates creation of higher complexity libraries) and the speed of FACS screens.

More recently, an academic laboratory has described a complementary strategy for the purpose of retargeting of meganuclease specificity. Well-characterized wild-type meganucleases are computationally screened to identify the best candidate protein to target a genomic region; that endonuclease is then redesigned via activity selections within compartmentalized aqueous droplets [126]. The use of this system allows the formation and interrogation of exceptionally large libraries of randomized endonuclease sequences (sampling up to 1010 constructs per selection step) as well as the tight control of temperature, time and concentration during individual selection steps over the course of endonuclease engineering. In this study, the method was illustrated by engineering several different meganucleases to cleave multiple human genomic sites, as well as variants that discriminate between single nucleotide polymorphism (SNP) variants. Simultaneous expression of two such fusion enzymes results in efficient excision of a defined genomic region (a property that, combined with the small size and coding sequences of homing endonucleases, is particularly useful for such applications).

(v) Refinement and extension of engineered homing endonuclease technologies

Beyond the development and demonstration of reliable methods for engineering homing endonuclease, their use as gene targeting reagents has been further facilitated by several recent developments. First, the number of wild-type homing endonucleases that have been identified and characterized has grown rapidly, along with the cataloguing and public deposition of their most important features of protein sequence, target sites, and structural features of recognition [127, 128]. Second, their unique ability to generate defined 3’ overhangs can enhance the recombinogenicity of their cleavage products, and also can be exploited for enhanced gene disruption through the parallel introduction of 3’ exonucleases [129, 130]. Third, their active sites are amenable to the introduction of individual point mutations, with the goal of generating site-specific nickase enzymes that can be used to control the outcome of competing repair pathways [131, 132]. Their compact size and the availability of free N- and C-termini has facilitated their fusion with auxiliary DNA targeting domains (in particular, through the addition of engineered TAL effector repeats) [125, 126] to create highly specific and active gene targeting nucleases that still comprise small, single chain, easily packaged scaffolds. Finally, extremely informative reporter systems and assays that allow precise measurements and quantitation of the mechanisms, efficiency, and repair pathway choice and outcome(s) resulting from nuclease-induced double-strand breaks have been developed [133135], facilitating the refinement and optimization of such systems for genome engineering applications.

Conclusions

There is much to be learned from the history of studies of mobile introns and their associated protein factors (which has the advantage, from the point of view of the investigator tasked with writing this review, of starting with an obvious ‘big-bang’ moment corresponding to their initial discovery in 1971). From a biological standpoint, perhaps the most important insights are those gained by reducing the complexities that surround the co-evolution of a host and a parasitic endosymbiont down to the simplest level: that of a DNA binding protein tasked with the recognition of an evolving genomic target. This simple molecular drama, conducted over many generations and replete with many nuances and subtleties, continues to play out in every biological kingdom, using all known types of homing endonucleases, split gene structures, and host genes. At the same time, the functional capabilities of these small mobile elements are continually being spun-off into new and different biological pathways and functions, ranging from the protection and maintenance of the genome (an ironic twist given that the fundamental purpose of a homing endonuclease is to act as an invasive element) to the transcriptional regulation of complex developmental processes.

Beyond these scientific points, the importance of homing endonucleases for genome engineering speaks clearly of the impact, often unexpected and unpredictable, that basic research, even of the most seemingly esoteric or mundane type, can have on the creation of entirely new areas of biotechnology and medicine. Just as studies of bacterial phage restriction in the early 1950s led to the discovery and application of restriction endonucleases (molecules that, when harnessed, paved the way for the creation and use of recombinant DNA and the establishment of the biotechnology industry), the study of seemingly minor and unimportant genetic markers in yeast and phage provided the initial steps into a field of targeted genetic modification and genome engineering that may revolutionize much of the way in which future biological studies are conducted.

Abbreviations

CRISPR:

clustered regularly interspaced short palindromic repeats

FACS:

flow-assisted cell sorting

MAT:

mating type

rDNA:

ribosomal DNA

TAL(EN):

transcriptional activator like (effector nuclease)

ZFN:

zinc-finger nuclease.

References

  1. Bos JL, Heyting C, Borst P, Arnberg AC, Van Bruggen EF: An insert in the single gene for the large ribosomal RNA in yeast mitochondrial DNA. Nature 1978, 275: 336-338. 10.1038/275336a0

    CAS  PubMed  Google Scholar 

  2. Faye G, Dennebouy N, Kujawa C, Jacq C: Inserted sequence in the mitochondrial 23S ribosomal RNA gene of the yeast saccharomyces cerevisiae. Mol Gen Genet 1979, 168: 101-109. 10.1007/BF00267939

    CAS  PubMed  Google Scholar 

  3. Bolotin M, Coen D, Deutsch J, Dujon B, Netter P, Petrochilo E, Slonimski PP: Recombination in mitochondria from Saccharomyces cerevisiae . Bull Inst Pasteur Paris 1971, 69: 215-239.

    Google Scholar 

  4. Dujon B: Sequence of the intron and flanking exons of the mitochondrial 21S rRNA gene of yeast strains having different alleles at the omega and rib-1 loci. Cell 1980, 20: 185-197. 10.1016/0092-8674(80)90246-9

    CAS  PubMed  Google Scholar 

  5. Lazowska J, Jacq C, Slonimski PP: Sequence of introns and flanking exons in wild-type and box3 mutants of cytochrome b reveals an interlaced splicing protein coded by an intron. Cell 1980, 22: 333-348. 10.1016/0092-8674(80)90344-X

    CAS  PubMed  Google Scholar 

  6. Klar A, Strathern J: Resolution of recombination intermediates generated during yeast mating type switching. Nature 1984, 310: 744-748. 10.1038/310744a0

    CAS  PubMed  Google Scholar 

  7. Kostriken R, Strathern JN, Klar AJ, Hicks JB, Heffron F: A site-specific endonuclease essential for mating-type switching in Saccharomyces cerevisiae. Cell 1983, 35: 167-174. 10.1016/0092-8674(83)90219-2

    CAS  PubMed  Google Scholar 

  8. Hensgens LAM, Bonen L, De Haan M, Horst G, Grivell LA: Two introns in yeast mitochondrial gene: homology among URF containing introns and strain dependent variation flanking exons. Cell 1983, 32: 379-389. 10.1016/0092-8674(83)90457-9

    CAS  PubMed  Google Scholar 

  9. Jacquier A, Dujon B: An intron-encoded protein is active in a gene conversion process that spreads an intron into a mitochondrial gene. Cell 1985, 41: 383-394. 10.1016/S0092-8674(85)80011-8

    CAS  PubMed  Google Scholar 

  10. Macreadie IG, Scott RM, Zinn AR, Butow RA: Transposition of an intron in yeast mitochondria requires a protein encoded by that intron. Cell 1985, 41: 395-402. 10.1016/S0092-8674(85)80012-X

    CAS  PubMed  Google Scholar 

  11. Zinn AR, Butow RA: Nonreciprocal exchange between alleles of the yeast mitochondrial 21S rRNA gene: kinetics and the involvement of a double-strand break. Cell 1985, 40: 887-895. 10.1016/0092-8674(85)90348-4

    CAS  PubMed  Google Scholar 

  12. Colleaux L, D'Auriol L, Betermier M, Cottarel G, Jacquier A, Galibert F, Dujon B: Universal code equivalent of a yeast mitochondrial intron reading frame is expressed into E. coli as a specific double strand endonuclease. Cell 1986, 44: 521-533. 10.1016/0092-8674(86)90262-X

    CAS  PubMed  Google Scholar 

  13. Colleaux L, D'Auriol L, Galibert F, Dujon B: Recognition and cleavage site of the intron-encoded omega transposase. Proc Natl Acad Sci U S A 1988, 85: 6022-6026. 10.1073/pnas.85.16.6022

    CAS  PubMed  PubMed Central  Google Scholar 

  14. Durrenberger F, Rochaix JD: Chloroplast ribosomal intron of Chlamydomonas reinhardtii: in vitro self-splicing, DNA endonuclease activity and in vivo mobility. Embo J 1991, 10: 3495-3501.

    CAS  PubMed  PubMed Central  Google Scholar 

  15. Remacle C, Matagne RF: Transmission, recombination and conversion of mitochondrial markers in relation to the mobility of a group I intron in Chlamydomonas. Curr Genet 1993, 23: 518-525. 10.1007/BF00312645

    CAS  PubMed  Google Scholar 

  16. Thompson AJ, Yuan X, Kudlicki W, Herrin DL: Cleavage and recognition pattern of a double-strand-specific endonuclease (I-creI) encoded by the chloroplast 23S rRNA intron of Chlamydomonas reinhardtii. Gene 1992, 119: 247-251. 10.1016/0378-1119(92)90278-W

    CAS  PubMed  Google Scholar 

  17. Lucas P, Otis C, Mercier JP, Turmel M, Lemieux C: Rapid evolution of the DNA-binding site in LAGLIDADG homing endonucleases. Nucleic Acids Res 2001, 29: 960-969. 10.1093/nar/29.4.960

    CAS  PubMed  PubMed Central  Google Scholar 

  18. Turmel M, Boulanger J, Schnare MN, Gray MW, Lemieux C: Six group I introns and three internal transcribed spacers in the chloroplast large subunit ribosomal RNA gene of the green alga Chlamydomonas eugametos. J Mol Biol 1991, 218: 293-311. 10.1016/0022-2836(91)90713-G

    CAS  PubMed  Google Scholar 

  19. Turmel M, Gutell RR, Mercier JP, Otis C, Lemieux C: Analysis of the chloroplast large subunit ribosomal RNA gene from 17 Chlamydomonas taxa. Three internal transcribed spacers and 12 group I intron insertion sites. J Mol Biol 1993, 232: 446-467. 10.1006/jmbi.1993.1402

    CAS  PubMed  Google Scholar 

  20. Kane P, Yamashiro C, Wolczyk D, Neff N, Goebl M, Stevens T: Protein splicing converts the yeast TFP1 gene product to the 69-kD subunit of the vacuolar H (+)-adenosine triphosphatase. Science 1990, 250: 651-657. 10.1126/science.2146742

    CAS  PubMed  Google Scholar 

  21. Gimble FS, Thorner J: Homing of a DNA endonuclease by meitoic gene conversion in Saccharomyces cerevisiae . Nature 1992, 357: 301-306. 10.1038/357301a0

    CAS  PubMed  Google Scholar 

  22. Gimble FS, Wang J: Substrate recognition and induced DNA distortion by the PI-SceI endonuclease, an enzyme generated by protein splicing. J Mol Biol 1996, 263: 163-180. 10.1006/jmbi.1996.0567

    CAS  PubMed  Google Scholar 

  23. Wende W, Grindl W, Christ F, Pingoud A, Pingoud V: Binding, bending and cleavage of DNA substrates by the homing endonuclease PI-SceI. Nucleic Acids Res 1996, 24: 4123-4132. 10.1093/nar/24.21.4123

    CAS  PubMed  PubMed Central  Google Scholar 

  24. Chu F, Maley G, Maley F, Belfort M: An intervening sequence in the thymidylate synthase gene of bacteriophage T4. Proc Natl Acad Sci U S A 1984, 81: 3149-3153.

    Google Scholar 

  25. Quirk SM, Bell-Pedersen D, Belfort M: Intron mobility in the T-even phages: high frequency inheritance of group I introns promoted by intron open reading frames. Cell 1989, 56: 455-465. 10.1016/0092-8674(89)90248-1

    CAS  PubMed  Google Scholar 

  26. Bell-Pedersen D, Quirk SM, Aubrey M, Belfort M: A site-specific endonuclease and co-conversion of flanking exons associated with the mobile td intron of phage T4. Gene 1989, 82: 119-126. 10.1016/0378-1119(89)90036-X

    CAS  PubMed  Google Scholar 

  27. Clyman J, Belfort M: Trans and cis requirements for intron mobility in a prokaryotic system. Genes Dev 1992, 6: 1269-1279. 10.1101/gad.6.7.1269

    CAS  PubMed  Google Scholar 

  28. Mueller JE, Clyman J, Huang YJ, Parker MM, Belfort M: Intron mobility in phage T4 occurs in the context of recombination-dependent DNA replication by way of multiple pathways. Genes Dev 1996, 10: 351-364. 10.1101/gad.10.3.351

    CAS  PubMed  Google Scholar 

  29. Belfort M, Reaban ME, Coetzee T, Dalgaard JZ: Prokaryotic introns and inteins: a panoply of form and function. J Bacter 1995, 177: 3897-3903.

    CAS  Google Scholar 

  30. Belfort M, Bonocora RP: Homing endonucleases: from genetic anomalies to programmable genomic clippers. Volume 1,123. In Homing Endonucleases: Methods and Protocols. Edited by: Edgell D. New York: Springer; Methods in Molecular Biology; 2014.

    Google Scholar 

  31. Michel F, Dujon B: Genetic exchanges between bacteriophage T4 and filamentous fungi. Cell 1986, 46: 323-330. 10.1016/0092-8674(86)90651-3

    CAS  PubMed  Google Scholar 

  32. Shub DA, Gott JM, Xu MQ, Lang BF, Michel F, Tomaschewski J, Pedersen-Lane J, Belfort M: Structural conservation among three homologous introns of bacteriophage T4 and the group I introns of eukaryotes. Proc Natl Acad Sci U S A 1988, 85: 1151-1155. 10.1073/pnas.85.4.1151

    CAS  PubMed  PubMed Central  Google Scholar 

  33. Goodrich-Blair H, Shub DA: Beyond homing: competition between intron endonucleases confers a selective advantage on flanking genetic markers. Cell 1996, 84: 211-221. 10.1016/S0092-8674(00)80976-9

    CAS  PubMed  Google Scholar 

  34. Turmel M, Cote V, Otis C, Mercier JP, Gray MW, Lonergan KM, Lemieux C: Evolutionary transfer of ORF-containing group I introns between different subcellular compartments (chloroplast and mitochondrion). Mol Biol Evol 1995, 12: 533-545.

    CAS  PubMed  Google Scholar 

  35. Cho Y, Qiu Y-L, Kuhlman P, Palmer JD: Explosive invasion of plant mitochondria by a group I intron. Proc Natl Acad Sci U S A 1998, 95: 14244-14249. 10.1073/pnas.95.24.14244

    CAS  PubMed  PubMed Central  Google Scholar 

  36. Edgell DR, Belfort M, Shub DA: Barriers to intron promiscuity in bacteria. J Bacteriology 2000, 182: 5281-5289. 10.1128/JB.182.19.5281-5289.2000

    CAS  Google Scholar 

  37. Edgell DR, Gibb EA, Belfort M: Mobile DNA elements in T4 and related phages: a review in the series on bacteriophage T4 and its relatives. Virol J 2010, 7: 290-300. 10.1186/1743-422X-7-290

    PubMed  PubMed Central  Google Scholar 

  38. Stoddard BL: Homing endonucleases: from microbial genetic invaders to reagents for targeted DNA modification. Structure 2011, 19: 7-15. 10.1016/j.str.2010.12.003

    CAS  PubMed  PubMed Central  Google Scholar 

  39. Bolduc JM, Spiegel PC, Chatterjee P, Brady KL, Downing ME, Caprara MG, Waring RB, Stoddard BL: Structural and biochemical analyses of DNA and RNA binding by a bifunctional homing endonuclease and group I intron splicing factor. Genes Dev 2003, 17: 2875-2888. 10.1101/gad.1109003

    CAS  PubMed  PubMed Central  Google Scholar 

  40. Duan X, Gimble FS, Quiocho FA: Crystal structure of PI-SceI, a homing endonuclease with protein splicing activity. Cell 1997, 89: 555-564. 10.1016/S0092-8674(00)80237-8

    CAS  PubMed  Google Scholar 

  41. Flick KE, Jurica MS, Monnat RJ Jr, Stoddard BL: DNA binding and cleavage by the nuclear intron-encoded homing endonuclease I-PpoI. Nature 1998, 394: 96-101. 10.1038/27952

    CAS  PubMed  Google Scholar 

  42. Heath PJ, Stephens KM, Monnat RJ, Stoddard BL: The structure of I- Cre I, a group I intron-encoded homing endonuclease. Nature Struct Biol 1997, 4: 468-476. 10.1038/nsb0697-468

    CAS  PubMed  Google Scholar 

  43. Jurica MS, Monnat RJ Jr, Stoddard BL: DNA recognition and cleavage by the LAGLIDADG homing endonuclease I-CreI. Mol Cell 1998, 2: 469-476. 10.1016/S1097-2765(00)80146-X

    CAS  PubMed  Google Scholar 

  44. Moure C, Gimble F, Quiocho F: Crystal structure of the intein homing endonuclease PI-SceI bound to its recognition sequence. Nature Struct Biol 2002, 9: 764-770. 10.1038/nsb840

    CAS  PubMed  Google Scholar 

  45. Moure CM, Gimble FS, Quiocho FA: The crystal structure of the gene targeting homing endonuclease I-SceI reveals the origins of its target site specificity. J Mol Biol 2003, 334: 685-696. 10.1016/j.jmb.2003.09.068

    CAS  PubMed  Google Scholar 

  46. Shen BW, Landthaler M, Shub DA, Stoddard BL: DNA binding and cleavage by the HNH homing endonuclease I-HmuI. J Mol Biol 2004, 342: 43-56. 10.1016/j.jmb.2004.07.032

    CAS  PubMed  Google Scholar 

  47. Taylor G, Heiter D, Pietrokovski S, Stoddard B: Activity, specificity and structure of I-Bth0305I: a representative of a new homing endonuclease family. Nucleic Acids Res 2011, 30: 9705-9719.

    Google Scholar 

  48. VanRoey P, Meehan L, Kowalski JC, Belfort M, Derbyshire V: Catalytic domain structure and hypothesis for function of GIY-YIG intron endonuclease I-TevI. Nat Struct Biol 2002, 9: 806-811.

    CAS  Google Scholar 

  49. VanRoey P, Waddling CA, Fox KM, Belfort M, Derbyshire V: Intertwined structure of the DNA-binding domain of intron endonuclease I-TevI with its substrate. EMBO J 2001, 20: 3631-3637. 10.1093/emboj/20.14.3631

    CAS  Google Scholar 

  50. Zhao L, Pellenz S, Stoddard BL: Activity and specificity of the bacterial PD-(D/E) XK homing endonuclease I-Ssp6803I. J Mol Biol 2008, 385: 1498-1510.

    PubMed  PubMed Central  Google Scholar 

  51. Sitbon E, Pietrokovski S: New types of conserved sequence domains in DNA-binding regions of homing endonucleases. Trends Biochem Sci 2003, 28: 473-477. 10.1016/S0968-0004(03)00170-1

    CAS  PubMed  Google Scholar 

  52. Muscarella DE, Vogt VM: A mobile group I intron in the nuclear rDNA of Physarum polycephalum . Cell 1989, 56: 443-454. 10.1016/0092-8674(89)90247-X

    CAS  PubMed  Google Scholar 

  53. Nomura N, Nomura Y, Sussman D, Klein D, Stoddard BL: Recognition of a common rDNA target site in archaea and eukarya by analogous LAGLIDADG and His-Cys box homing endonucleases. Nucleic Acids Res 2008, 36: 6988-6998. 10.1093/nar/gkn846

    CAS  PubMed  PubMed Central  Google Scholar 

  54. Jakubczak JL, Burke WD, Eickbush TH: Retrotransposable elements R1 and R2 interrupt the rRNA genes of most insects. Proc Natl Acad Sci U S A 1991, 88: 3295-3299. 10.1073/pnas.88.8.3295

    CAS  PubMed  PubMed Central  Google Scholar 

  55. Chevalier B, Turmel M, Lemieux C, Monnat RJ, Stoddard BL: Flexible DNA target site recognition by divergent homing endonuclease isoschizomers I-CreI and I-MsoI. J Mol Biol 2003, 329: 253-269. 10.1016/S0022-2836(03)00447-9

    CAS  PubMed  Google Scholar 

  56. Edgell DR, Stanger MJ, Belfort M: Importance of a single base pair for discrimination between intron-containing and intron-less alleles by endonuclease I-BmoI. Curr Biol 2003, 13: 973-978. 10.1016/S0960-9822(03)00340-3

    CAS  PubMed  Google Scholar 

  57. Edgell DR, Stanger MJ, Belfort M: Coincidence of cleavage sites of intron endonuclease I-TevI and critical sequences of the host thymidylate synthase gene. J Mol Biol 2004, 343: 1231-1241. 10.1016/j.jmb.2004.09.005

    CAS  PubMed  Google Scholar 

  58. Landthaler M, Shen BW, Stoddard BL, Shub DA: I-BasI and I-HmuI: two phage intron-encoded endonucleases with homologous DNA recognition sequences but distinct DNA specificities. J Mol Biol 2006, 358: 1137-1151. 10.1016/j.jmb.2006.02.054

    CAS  PubMed  Google Scholar 

  59. Argast GM, Stephens KM, Emond MJ, Monnat RJ: I- Ppo I and I- Cre I homing site sequence degeneracy determined by random mutagenesis and sequential in vitro enrichment. J Mol Biol 1998, 280: 345-353. 10.1006/jmbi.1998.1886

    CAS  PubMed  Google Scholar 

  60. Gimble FS, Moure CM, Posey KL: Assessing the plasticity of DNA target site recognition of the PI-SceI homing endonuclease using a bacterial two-hybrid selection system. J Mol Biol 2003, 334: 993-1008. 10.1016/j.jmb.2003.10.013

    CAS  PubMed  Google Scholar 

  61. Perrin A, Buckle M, Dujon B: Asymmetrical recognition and activity of the I-SceI endonuclease on its site and on intron-exon junctions. EMBO J 1993, 12: 2939-2947.

    CAS  PubMed  PubMed Central  Google Scholar 

  62. Scalley-Kim M, McConnell-Smith A, Stoddard BL: Coevolution of homing endonuclease specificity and its host target sequence. J Mol Biol 2007, 372: 1305-1319. 10.1016/j.jmb.2007.07.052

    CAS  PubMed  PubMed Central  Google Scholar 

  63. Jarjour J, West-Foyle H, Certo MT, Hubert CG, Doyle L, Getz MM, Stoddard BL, Scharenberg AM: High-resolution profiling of homing endonuclease binding and catalytic specificity using yeast surface display. Nucleic Acids Res 2009, 37: 6871-6880. 10.1093/nar/gkp726

    CAS  PubMed  PubMed Central  Google Scholar 

  64. Thyme SB, Jarjour J, Takeuchi R, Havranek JJ, Ashworth J, Scharenberg AM, Stoddard BL, Baker D: Exploitation of binding energy for catalysis and design. Nature 2009, 461: 1300-1304. 10.1038/nature08508

    CAS  PubMed  PubMed Central  Google Scholar 

  65. Petek LM, Russell DW, Miller DG: Frequent endonuclease cleavage at off-target locations in vivo. Mol Ther 2010, 18: 983-986. 10.1038/mt.2010.35

    CAS  PubMed  PubMed Central  Google Scholar 

  66. Yang W, Lee JY, Nowotny M: Making and breaking nucleic acids: two-Mg2 + −ion catalysis and substrate specificity. Mol Cell 2006, 22: 5-13. 10.1016/j.molcel.2006.03.013

    CAS  PubMed  Google Scholar 

  67. Chevalier B, Sussman D, Otis C, Noel AJ, Turmel M, Lemieux C, Stephens K, Monnat RJ Jr, Stoddard BL: Metal-dependent DNA cleavage mechanism of the I-CreI LAGLIDADG homing endonuclease. Biochemistry 2004, 43: 14015-14026. 10.1021/bi048970c

    CAS  PubMed  Google Scholar 

  68. Zhao L, Bonocora RP, Shub DA, Stoddard BL: The restriction fold turns to the dark side: a bacterial homing endonuclease with a PD-(D/E)-XK motif. Embo J 2007, 26: 2432-2442. 10.1038/sj.emboj.7601672

    CAS  PubMed  PubMed Central  Google Scholar 

  69. Chevalier BS, Monnat RJ Jr, Stoddard BL: The homing endonuclease I-CreI uses three metals, one of which is shared between the two active sites. Nat Struct Biol 2001, 8: 312-316. 10.1038/86181

    CAS  PubMed  Google Scholar 

  70. Galburt EA, Chevalier B, Tang W, Jurica MS, Flick KE, Monnat RJ, Stoddard BL: A novel endonuclease mechanism directly visualized for I-PpoI. Nat Struct Biol 1999, 6: 1096-1099. 10.1038/70027

    CAS  PubMed  Google Scholar 

  71. Burt A, Koufopanou V: Homing endonuclease genes: the rise and fall and rise again of a selfish element. Curr Opin Gen Dev 2004, 14: 609-615. 10.1016/j.gde.2004.09.010

    CAS  Google Scholar 

  72. Delahodde A, Goguel V, Becam AM, Creusot F, Perea J, Banroques J, Jacq C: Site-specific DNA endonuclease and RNA maturase activities of two homologous intron-encoded proteins from yeast mitochondria. Cell 1989, 56: 431-441. 10.1016/0092-8674(89)90246-8

    CAS  PubMed  Google Scholar 

  73. Dujardin G, Jacq C, Slonimski PP: Single base substitution in an intron of oxidase gene compensates splicing defects of the cytochrome b gene. Nature 1982, 298: 628-632. 10.1038/298628a0

    CAS  PubMed  Google Scholar 

  74. Chatterjee P, Brady KL, Solem A, Ho Y, Caprara MG: Functionally distinct nucleic acid binding sites for a group I intron-encoded RNA maturase/DNA homing endonuclease. J Mol Biol 2003, 329: 239-251. 10.1016/S0022-2836(03)00426-1

    CAS  PubMed  Google Scholar 

  75. Edgell DR, Derbyshire V, Van Roey P, LaBonne S, Stanger MJ, Li Z, Boyd TM, Shub DA, Belfort M: Intron-encoded homing endonuclease I-TevI also functions as a transcriptional autorepressor. Nat Struct Mol Biol 2004, 11: 936-944. 10.1038/nsmb823

    CAS  PubMed  Google Scholar 

  76. Taylor G, Stoddard B: Structural, functional and evolutionary relationships between homing endonucleases and proteins from their host organisms. Nucleic Acids Res 2012, 40: 189-200.

    Google Scholar 

  77. Segal DJ, Meckler JF: Genome engineering at the dawn of the golden age. Ann Rev Genomics Hum Genet 2013. doi:10.1146/annurev-genom-091212-153435

    Google Scholar 

  78. Choulika A, Perrin A, Dujon B, Nicolas JF: Induction of homologous recombination in mammalian chromosomes by using the I-SceI system of Saccharomyces cerevisiae. Mol Cell Biol 1968–1973, 1995: 15.

    Google Scholar 

  79. 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.

    CAS  PubMed  PubMed Central  Google Scholar 

  80. Rouet P, Smih F, Jasin M: Expression of a site-specific endonuclease stimulates homologous recombination in mammalian cells. Proc Natl Acad Sci U S A 1994, 91: 6064-6068. 10.1073/pnas.91.13.6064

    CAS  PubMed  PubMed Central  Google Scholar 

  81. Kim YG, Cha J, Chandrasegaran S: Hybrid restriction enzymes: zinc finger fusions to Fok I cleavage domain. Proc Natl Acad Sci U S A 1996, 93: 1156-1160. 10.1073/pnas.93.3.1156

    CAS  PubMed  PubMed Central  Google Scholar 

  82. Smith J, Bibikova M, Whitby FG, Reddy AR, Chandrasegaran S, Carroll D: Requirements for double-strand cleavage by chimeric restriction enzymes with zinc finger DNA-recognition domains. Nucleic Acids Res 2000, 28: 3361-3369. 10.1093/nar/28.17.3361

    CAS  PubMed  PubMed Central  Google Scholar 

  83. Bibikova M, Golic M, Golic KG, Carroll D: Targeted chromosomal cleavage and mutagenesis in Drosophila using zinc-finger nucleases. Genetics 2002, 161: 1169-1175.

    CAS  PubMed  PubMed Central  Google Scholar 

  84. Christian M, Cermak T, Doyle EL, Schmidt C, Zhang F, Hummel A, Bogdanove AJ, Voytas DF: Targeting DNA double-strand breaks with TAL effector nucleases. Genetics 2010, 186: 757-761. 10.1534/genetics.110.120717

    CAS  PubMed  PubMed Central  Google Scholar 

  85. Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N, Hsu PD, Wu X, Jiang W, Marraffini LA, Zhang F: Multiplex genome engineering using CRISPR/Cas systems. Science 2013, 339: 819-823. 10.1126/science.1231143

    CAS  PubMed  PubMed Central  Google Scholar 

  86. Mali P, Yang L, Esvelt KM, Aach J, Guell M, DiCarlo JE, Norville JE, Church GM: RNA-guided human genome engineering via Cas9. Science 2013, 339: 823-826. 10.1126/science.1232033

    CAS  PubMed  PubMed Central  Google Scholar 

  87. Ran FA, Hsu PD, Lin CY, Gootenberg JS, Konermann S, Trevino AE, Scott DA, Inoue A, Matoba S, Zhang Y, Zhang F: Double nicking by RNA-guided CRISPR Cas9 for enhanced genome editing specificity. Cell 2013, 154: 1380-1389. 10.1016/j.cell.2013.08.021

    CAS  PubMed  PubMed Central  Google Scholar 

  88. Arnould S, Perez C, Cabaniols J-P, Smith J, Gouble A, Grizot S, Epinat J-C, Duclert A, Duchateau P, Paques F: Engineered I-CreI derivatives cleaving sequences from the human XPC gene can induce highly efficient gene correction in mammalian cells. J Mol Biol 2007, 371: 49-65. 10.1016/j.jmb.2007.04.079

    CAS  PubMed  Google Scholar 

  89. Pennisi E: The CRISPR craze. Science 2013, 341: 833-836. 10.1126/science.341.6148.833

    CAS  PubMed  Google Scholar 

  90. Kleinstiver BP, Wolfs JM, Kolaczyk T, Roberts AK, Hu SX, Edgell DR: Monomeric site-specific nucleases for genome editing. Proc Natl Acad Sci U S A 2012, 109: 8061-8066. 10.1073/pnas.1117984109

    CAS  PubMed  PubMed Central  Google Scholar 

  91. Schierling B, Dannemann N, Gabsalilow L, Wende W, Cathomen T, Pingoud A: A novel zinc-finger nuclease platform with a sequence-specific cleavage module. Nucleic Acids Res 2012, 40: 2623-2638. 10.1093/nar/gkr1112

    CAS  PubMed  PubMed Central  Google Scholar 

  92. Yanik M, Alzubi J, Lahaye T, Cathomen T, Pingoud A, Wende W: TALE-PvuII fusion proteins - novel tools for gene targeting. PLoS One 2013, 8: e82539. 10.1371/journal.pone.0082539

    PubMed  PubMed Central  Google Scholar 

  93. Flick KE, McHugh D, Heath JD, Stephens KM, Monnat RJ Jr, Stoddard BL: Crystallization and preliminary X-ray studies of I-PpoI: a nuclear, intron-encoded homing endonuclease from Physarum polycephalum. Protein Sci 1997, 6: 2677-2680.

    CAS  PubMed  PubMed Central  Google Scholar 

  94. Seligman L, Chisholm KM, Chevalier BS, Chadsey MS, Edwards ST, Savage JH, Veillet AL: Mutations altering the cleavage specificity of a homing endonuclease. Nucleic Acids Res 2002, 30: 3870-3879. 10.1093/nar/gkf495

    CAS  PubMed  PubMed Central  Google Scholar 

  95. Gruen M, Chang K, Serbanescu I, Liu DR: An in vivo selection system for homing endonuclease activity. Nucleic Acids Res 2002, 30: 29-34. 10.1093/nar/30.7.e29

    Google Scholar 

  96. Ashworth J, Havranek JJ, Duarte CM, Sussman D, Monnat RJ Jr, Stoddard BL, Baker D: Computational redesign of endonuclease DNA binding and cleavage specificity. Nature 2006, 441: 656-659. 10.1038/nature04818

    CAS  PubMed  PubMed Central  Google Scholar 

  97. Sussman DJ, Chadsey M, Fauce S, Engel A, Bruett A, RJMonnat J, Stoddard BL, Seligman LM: Isolation and characterization of new homing endonuclease specificities at individual target site positions. J Mol Biol 2004, 342: 31-41. 10.1016/j.jmb.2004.07.031

    CAS  PubMed  Google Scholar 

  98. Chames P, Epinat JC, Guillier S, Patin A, Lacroix E, Paques F: In vivo selection of engineered homing endonucleases using double-strand break induced homologous recombination. Nucleic Acids Res 2005, 33: e178. 10.1093/nar/gni175

    PubMed  PubMed Central  Google Scholar 

  99. Smith J, Grizot S, Arnould S, Duclert A, Epinat JC, Chames P, Prieto J, Redondo P, Blanco FJ, Bravo J, Montoya G, Paques F, Duchateau P: A combinatorial approach to create artificial homing endonucleases cleaving chosen sequences. Nucleic Acids Res 2006, 34: e149. 10.1093/nar/gkl720

    PubMed  PubMed Central  Google Scholar 

  100. Arnould S, Chames P, Perez C, Lacroix E, Duclert A, Epinat JC, Stricher F, Petit AS, Patin A, Guillier S, Rolland S, Prieto J, Blanco FJ, Bravo J, Montoya G, Serrano L, Duchateau P, Paques F: Engineering of large numbers of highly specific homing endonucleases that induce recombination on novel DNA targets. J Mol Biol 2006, 355: 443-458. 10.1016/j.jmb.2005.10.065

    CAS  PubMed  Google Scholar 

  101. Paques F, Duchateau P: Meganucleases and DNA double-strand break-induced recombination: perspectives for gene therapy. Curr Gene Ther 2007, 7: 49-66. 10.2174/156652307779940216

    CAS  PubMed  Google Scholar 

  102. Ashworth J, Taylor GK, Havranek JJ, Quadri SA, Stoddard BL, Baker D: Computational reprogramming of homing endonuclease specificity at multiple adjacent base pairs. Nucleic Acids Res 2010, 38: 5601-5608. 10.1093/nar/gkq283

    CAS  PubMed  PubMed Central  Google Scholar 

  103. Steuer S, Pingoud V, Pingoud A, Wende W: Chimeras of the homing endonuclease PI-SceI and the homologous candida tropicalis intein: a study to explore the possibility of exchanging DNA-binding modules to obtain highly speciric endonucleases with altered specificity. Chembiochem 2004, 5: 206-213. 10.1002/cbic.200300718

    CAS  PubMed  Google Scholar 

  104. Epinat JC, Arnould S, Chames P, Rochaix P, Desfontaines D, Puzin C, Patin A, Zanghellini A, Paques F, Lacroix E: A novel engineered meganuclease induces homologous recombination in yeast and mammalian cells. Nucleic Acids Res 2003, 31: 2952-2962. 10.1093/nar/gkg375

    CAS  PubMed  PubMed Central  Google Scholar 

  105. Chevalier BS, Kortemme T, Chadsey MS, Baker D, RJMonnat J, Stoddard BL: Design, activity and structure of a highly specific artificial endonuclease. Molec Cell 2002, 10: 895-905. 10.1016/S1097-2765(02)00690-1

    CAS  PubMed  Google Scholar 

  106. Fitzsimons-Hall M, Noren CJ, Perler FB, Schildkraut I: Creation of an artificial bifunctional intein by grafting a homing endonuclease into a mini-intein. J Mol Biol 2002, 323: 173-179. 10.1016/S0022-2836(02)00912-9

    CAS  PubMed  Google Scholar 

  107. Silva GH, Belfort M: Analysis of the LAGLIDADG interface of the monomeric homing endonuclease I-DmoI. Nucleic Acids Res 2004, 32: 3156-3168. 10.1093/nar/gkh618

    CAS  PubMed  PubMed Central  Google Scholar 

  108. Silva GH, Belfort M, Wende W, Pingoud A: From monomeric to homodimeric endonucleases and back: engineering novel specificity of LAGLIDADG enzymes. J Mol Biol 2006, 361: 744-754. 10.1016/j.jmb.2006.06.063

    CAS  PubMed  Google Scholar 

  109. Baxter S, Lambert AR, Kuhar R, Jarjour J, Kulshina N, Parmeggiani F, Danaher P, Gano J, Baker D, Stoddard BL, Scharenberg AM: Engineering domain fusion chimeras from I-OnuI family LAGLIDADG homing endonucleases. Nucleic Acids Res 2012, 40: 7985-8000. 10.1093/nar/gks502

    CAS  PubMed  PubMed Central  Google Scholar 

  110. Gao H, Smith J, Yang M, Jones S, Djukanovic V, Nicholson MG, West A, Bidney D, Falco SC, Jantz D, Lyznik LA: Heritable targeted mutagenesis in maize using a designed endonuclease. Plant J 2010, 61: 176-187. 10.1111/j.1365-313X.2009.04041.x

    CAS  PubMed  Google Scholar 

  111. Grizot S, Smith J, Daboussi F, Prieto J, Redondo P, Merino N, Villate M, Thomas S, Lemaire L, Montoya G, Blanco FJ, Paques F, Duchateau P: Efficient targeting of a SCID gene by an engineered single-chain homing endonuclease. Nucleic Acids Res 2009, 37: 5405-5419. 10.1093/nar/gkp548

    CAS  PubMed  PubMed Central  Google Scholar 

  112. Li H, Pellenz S, Ulge U, Stoddard BL, Monnat RJ Jr: Generation of single-chain LAGLIDADG homing endonucleases from native homodimeric precursor proteins. Nucleic Acids Res 2009, 37: 1650-1662. 10.1093/nar/gkp004

    CAS  PubMed  PubMed Central  Google Scholar 

  113. Redondo P, Prieto J, Munoz IG, Alibes A, Stricher F, Serrano L, Cabaniols JP, Daboussi F, Arnould S, Perez C, Duchateau P, Paques F, Blanco FJ, Montoya G: Molecular basis of xeroderma pigmentosum group C DNA recognition by engineered meganucleases. Nature 2008, 456: 107-111. 10.1038/nature07343

    CAS  PubMed  Google Scholar 

  114. Dupuy A, Valton J, Leduc S, Armier J, Galetto R, Gouble A, Lebuhotel C, Stary A, Paques F, Duchateau P: Targeted gene therapy of xeroderma pigmentosum cells using meganuclease and TALEN. PLoS One 2013, 8: e78678. 10.1371/journal.pone.0078678

    PubMed  PubMed Central  Google Scholar 

  115. Cabaniols JP, Ouvry C, Lamamy V, Fery I, Craplet ML, Moulharat N, Guenin SP, Bedut S, Nosjean O, Ferry G, Devavry S, Jacqmarcq C, Lebuhotel C, Mathis L, Delenda C, Boutin JA, Duchateau P, Coge F, Paques F: Meganuclease-driven targeted integration in CHO-K1 cells for the fast generation of HTS-compatible cell-based assays. J Biomol Screen 2010, 15: 956-967. 10.1177/1087057110375115

    CAS  PubMed  Google Scholar 

  116. Cabaniols JP, Paques F: Robust cell line development using meganucleases. Methods Mol Biol 2008, 435: 31-45. 10.1007/978-1-59745-232-8_3

    CAS  PubMed  Google Scholar 

  117. Djukanovic V, Smith J, Lowe K, Yang M, Gao H, Jones S, Nicholson MG, West A, Lape J, Bidney D, Carl Falco S, Jantz D, Alexander Lyznik L: Male-sterile maize plants produced by targeted mutagenesis of the cytochrome P450-like gene (MS26) using a re-designed I-CreI homing endonuclease. Plant J 2013, 76: 888-899. 10.1111/tpj.12335

    CAS  PubMed  Google Scholar 

  118. Antunes MS, Smith JJ, Jantz D, Medford JI: Targeted DNA excision in Arabidopsis by a re-engineered homing endonuclease. BMC Biotechnol 2012, 12: 86. 10.1186/1472-6750-12-86

    CAS  PubMed  PubMed Central  Google Scholar 

  119. D'Halluin K, Vanderstraeten C, Van Hulle J, Rosolowska J, Van Den Brande I, Pennewaert A, D'Hont K, Bossut M, Jantz D, Ruiter R, Broadhvest J: Targeted molecular trait stacking in cotton through targeted double-strand break induction. Plant Biotechnol J 2013, 11: 933-941. 10.1111/pbi.12085

    PubMed  PubMed Central  Google Scholar 

  120. Munoz IG, Prieto J, Subramanian S, Coloma J, Redondo P, Villate M, Merino N, Marenchino M, D'Abramo M, Gervasio FL, Grizot S, Daboussi F, Smith J, Chion-Sotinel I, Paques F, Duchateau P, Alibes A, Stricher F, Serrano L, Blanco FJ, Montoya G: Molecular basis of engineered meganuclease targeting of the endogenous human RAG1 locus. Nucleic Acids Res 2011, 39: 729-743. 10.1093/nar/gkq801

    CAS  PubMed  PubMed Central  Google Scholar 

  121. Menoret S, Fontaniere S, Jantz D, Tesson L, Thinard R, Remy S, Usal C, Ouisse LH, Fraichard A, Anegon I: Generation of Rag1-knockout immunodeficient rats and mice using engineered meganucleases. FASEB J 2013, 27: 703-711. 10.1096/fj.12-219907

    CAS  PubMed  Google Scholar 

  122. Grosse S, Huot N, Mahiet C, Arnould S, Barradeau S, Clerre DL, Chion-Sotinel I, Jacqmarcq C, Chapellier B, Ergani A, Desseaux C, Cedrone F, Conseiller E, Paques F, Labetoulle M, Smith J: Meganuclease-mediated inhibition of HSV1 infection in cultured cells. Mol Ther 2011, 19: 694-702. 10.1038/mt.2010.302

    CAS  PubMed  PubMed Central  Google Scholar 

  123. Popplewell L, Koo T, Leclerc X, Duclert A, Mamchaoui K, Gouble A, Mouly V, Voit T, Paques F, Cedrone F, Isman O, Yanez-Munoz RJ, Dickson G: Gene correction of a duchenne muscular dystrophy mutation by meganuclease-enhanced exon knock-in. Hum Gene Ther 2013, 24: 692-701. 10.1089/hum.2013.081

    CAS  PubMed  Google Scholar 

  124. Chan YS, Takeuchi R, Jarjour J, Huen DS, Stoddard BL, Russell S: The design and in vivo evaluation of engineered I-OnuI-based enzymes for HEG gene drive. PLoS One 2013, 8: e74254. 10.1371/journal.pone.0074254

    CAS  PubMed  PubMed Central  Google Scholar 

  125. Boissel SJ, Astrakhan A, Jarjour J, Adey A, Shendure J, Stoddard BL, Certo M, Baker D, Scharenberg AM: MegaTALs: a rare-cleaving nuclease architecture for therapeutic genome engineering. Nucleic Acids Res 2013. Epub ahead of print (26 Nov 2013) doi:10.1093/nar/gkt1224

    Google Scholar 

  126. Takeuchi R, Choi M, Stoddard BL: Efficient engineering of meganucleases and MegaTALs using bioinformatics and in vitro compartmentalization for targeted gene modification. PNAS USA 2014. in press

    Google Scholar 

  127. Barzel A, Privman E, Peeri M, Naor A, Shachar E, Burstein D, Lazary R, Gophna U, Pupko T, Kupiec M: Native homing endonucleases can target conserved genes in humans and in animal models. Nucleic Acids Res 2011, 39: 6646-6659. 10.1093/nar/gkr242

    CAS  PubMed  PubMed Central  Google Scholar 

  128. Taylor GK, Petrucci LH, Lambert AR, Baxter SK, Jarjour J, Stoddard BL: LAHEDES: the LAGLIDADG homing endonuclease database and engineering server. Nucleic Acids Res 2012. (Webserver Issue) W110-116

    Google Scholar 

  129. Certo MT, Gwiazda KS, Kuhar R, Sather B, Curinga G, Mandt T, Brault M, Lambert AR, Baxter SK, Jacoby K, Ryu BY, Kiem HP, Gouble A, Paques F, Rawlings DJ, Scharenberg AM: Coupling endonucleases with DNA end-processing enzymes to drive gene disruption. Nat Methods 2012, 9: 973-975. 10.1038/nmeth.2177

    CAS  PubMed  PubMed Central  Google Scholar 

  130. Duchateau P, Delacote F, Perez C, Guyot V, Duhamel M, Rochon C, Ollivier N, Macmaster R, Silva GH, Paques F, Daboussi F: High frequency targeted mutagenesis using engineered endonucleases and DNA-end processing enzymes. PLoS One 2013, 8: e53217-e53225. 10.1371/journal.pone.0053217

    PubMed  PubMed Central  Google Scholar 

  131. McConnell Smith A, Takeuchi R, Pellenz S, Davis L, Maizels N, Monnat RJ Jr, Stoddard BL: Generation of a nicking enzyme that stimulates site-specific gene conversion from the I-AniI LAGLIDADG homing endonuclease. Proc Natl Acad Sci U S A 2009, 106: 5099-5104. 10.1073/pnas.0810588106

    CAS  PubMed  PubMed Central  Google Scholar 

  132. Metzger MJ, McConnell-Smith A, Stoddard BL, Miller AD: Single-strand nicks induce homologous recombination with less toxicity than double-strand breaks using an AAV template. Nucleic Acids Res 2010, 39: 926-935.

    PubMed  PubMed Central  Google Scholar 

  133. Certo MT, Ryu BY, Annis JE, Garibov M, Jarjour J, Rawlings DJ, Scharenberg AM: Tracking genome engineering outcome at individual DNA breakpoints. Nat Methods 2011, 8: 671-676. 10.1038/nmeth.1648

    CAS  PubMed  PubMed Central  Google Scholar 

  134. Daboussi F, Zaslavskiy M, Poirot L, Loperfido M, Gouble A, Guyot V, Leduc S, Galetto R, Grizot S, Oficjalska D, Perez C, Delacôte F, Dupuy A, Chion-Sotinel I, Le Clerre D, Lebuhotel C, Danos O, Lemaire F, Oussedik K, Cédrone F, Epinat JC, Smith J, Yáñez-Muñoz RJ, Dickson G, Popplewell L, Koo T, VandenDriessche T, Chuah MK, Duclert A, Duchateau P, et al.: Chromosomal context and epigenetic mechanisms control the efficacy of genome editing by rare-cutting designer endonucleases. Nucleic Acids Res 2012, 40: 6367-6379. 10.1093/nar/gks268

    CAS  PubMed  PubMed Central  Google Scholar 

  135. Kuhar R, Gwiazda KS, Humbert O, Mandt T, Pangallo J, Brault M, Khan I, Maizels N, Rawlings DJ, Scharenberg AM, Certo MT: Novel fluorescent genome editing reporters for monitoring DNA repair pathway utilization at endonuclease-induced breaks. Nucleic Acids Res 2013. epub ahead of print (10 Oct) doi:10.1093/nar/gkt872

    Google Scholar 

Download references

Acknowledgments

The work conducted in the authors laboratory, and the preparation of this review, were supported by a research grant from the NIH (R01 GM49857). The author thanks Dr Marlene Belfort for helpful suggestions and critiques.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Barry L Stoddard.

Additional information

Competing interests

BLS is a founder and shareholder in a private biotechnology company (Pregenen Inc.) that creates and uses engineered homing endonucleases for genomic and cellular engineering applications.

Authors’ original submitted files for images

Rights and permissions

This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Reprints and permissions

About this article

Cite this article

Stoddard, B.L. Homing endonucleases from mobile group I introns: discovery to genome engineering. Mobile DNA 5, 7 (2014). https://doi.org/10.1186/1759-8753-5-7

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1186/1759-8753-5-7

Keywords