Engineered Meganucleases/Homing Endonucleases

Meganucleases are sequence-specific endonucleases that use large (414 bp) recognition sites to generate accurate double-strand breaks (DSBs), promoting efficient gene targeting through homologous recombination (HR). Their recognition sites are extremely rare, and in a mammalian-sized genome, only a few such sites will exist. Accordingly, these proteins are perfect tools for genome editing as they are specific enough to bind to and cut only one site in a selected genome. In this sense, members of this enzyme family can be used as engineer tools that cleave DNA sequences other than their original wild-type targets, stimulating DSB-HR to induce the repair of defective genes in cells with a very low level of toxicity.

In terms of homing endonucleases (HEs), they can be used to actively induce HR following the induction of a DSB into the DNA. HEs can induce HR in different cell types, including mammalian and human cells, and they are useful to develop customized tools for gene targeting. HEs may have many potential biomedical and biotechnological applications, ranging from genome to gene therapy.

Introduction

HEs are small proteins (< 300 amino acids) found in bacteria, archaea, and in unicellular eukaryotes. A distinguishing characteristic of HEs is that they recognize relatively long sequences (14-40 bp) compared to other site-specific endonucleases such as restriction enzymes (4-8 bp). These lengthy recognition sites, and the name of the first such known enzyme, ω (also known as I-SceI), have given rise to the term "meganuclease". HEs are double-stranded DNases that have large, asymmetric recognition sites and coding sequences that are usually embedded in either introns or inteins. Introns are spliced out of precursor RNAs, while inteins are spliced out of precursor proteins.

HEs are highly specific and have evolved to cleave target sequences within cognate alleles without being overly toxic to the organism. They tolerate some individual base variation at their homing site, which ensures their propagation despite the evolutionary drift of their target sequence. Finally, HEs tend to be small proteins of <40 kDa, a property likely due to length limitations of the mobile sequences in which they reside.

Mechanism of HEs

The DNA binding domain of these proteins displays little modularity, with only two clearly distinct DNA-binding subdomains. Redesign of the DNA-binding interface depends on the identification of smaller subunits, which are far from being independent. The catalytic center (red) is embedded in the DNA binding interface (Fig.1).

Figure 1. Meganucleases and engineered meganucleases. (Galetto, 2009)

HEs Families

HEs families have been grouped into five families based on their sequence and structural motifs: LADGLIDADG, HNH, GIY-YIG, His-Cys box and PD-(D/E)XK.

Figure 2. Five families of HEs are shown with examples indicated in parenthesis. (Belfort, 2014)

• The LAGLIDADG endonucleases (LHE)

The LAGLIDADG endonucleases (LHE) make up a large, well-described family of HEs identified largely in archaea and organelles of lower eukaryotes. LHE genes exist in a variety of genomic environments, being encoded within group I introns, archaeal rRNA introns, as in-frame fusions with inteins, and as freestanding genes.

• GIY-YIG endonucleases

GIY-YIG endonucleases are modular proteins that exist in all three domains of life, including the Archaea, the Bacteria, and the Eukarya. In addition to GIY-YIG motifs in some restriction enzymes and association with the reverse transcriptase of Penelope retroelements, the motif also occurs in enzymes involved in DNA repair and maintenance of chromosomal genome stability. These include eukaryotic flap endonucleases, eukaryotic S1×1-S1×4 resolvase, and bacterial UvrC nucleotide excision repair protein.

• HNH Family

Members of the HNH family have been found in group I introns in plastids and phages, group II introns, and free-standing ORFs of bacteria, phages, and a cyanobacterial intein. The HNH motif consists of a stretch of approximately 30 amino acids that contain three highly conserved histidines and/or aspargine residues. Structural analysis indicates that the HNH family plays a role in metal binding for the first two conserved amino acids.

• His-Cys box homing endonucleases

The His-Cys box homing endonucleases comprise a much smaller family of HEs, found in group I introns interrupting nuclear rRNA genes of lower eukaryotes. As the name implies, the signature of this family is a region that is rich in histidine and cysteine residues. The best-studied of the His-Cys Box family is I-PpoI, a group I intron-encoded endonuclease from the slime mold Physarum polycephalum.

• PD-(D/E)xK and ED×HD Endonucleases

Restriction endonucleases share very little sequence conservation; however many contain a common catalytic fold from the PD-(D/E)xK superfamily. This motif appears to have been adapted by the group I intron-encoded homing endonuclease I-Ssp6803I from the cyanobacterium Synechocystis sp. 6803. I-Ssp6803I is a small protein that functions as a tetramer to recognize a large target site. Like other HEs, this recognition involves a paucity of protein-DNA contacts utilizing only one-third of the possible hydrogen bonds. This contrasts with restriction endonucleases which use a high density of protein-DNA contacts to recognize small DNA target sites.

Applications of HEs

HEs, which allow insertion, deletion, single-site mutation, and correction, are in a highly site-specific and controlled fashion. HEs can be used in the following fields:

• HEs as Therapeutic Agents
• HEs in Insect Vector Control
• HEs in Agriculture

References

1. Galetto, R.; et al. (2009). Targeted approaches for gene therapy and the emergence of engineered meganucleases. Expert opinion on biological therapy. 9(10): 1289-1303.
2. Belfort, M.; Bonocora, R. P. (2014). Homing endonucleases: from genetic anomalies to programmable genomic clippers. In Homing Endonucleases. 1-26.