Programmable Genome Editing Tools and their Regulation for Efficient Genome Engineering.

Targeted genome editing has become a powerful genetic tool for studying gene function or for modifying genomes by correcting defective genes or introducing genes. A variety of reagents have been developed in recent years that can generate targeted double-stranded DNA cuts which can be repaired by the error-prone, non-homologous end joining repair system or via the homologous recombination-based double-strand break repair pathway provided a suitable template is available. These genome editing reagents require components for recognizing a specific DNA target site and for DNA-cleavage that generates the double-stranded break. In order to reduce potential toxic effects of genome editing reagents, it might be desirable to control the in vitro or in vivo activity of these reagents by incorporating regulatory switches that can reduce off-target activities and/or allow for these reagents to be turned on or off. This review will outline the various genome editing tools that are currently available and describe the strategies that have so far been employed for regulating these editing reagents. In addition, this review will examine potential regulatory switches/strategies that can be employed in the future in order to provide temporal control for these reagents.

Insertion of Group II Intron-Based Ribozyme Switches into Homing Endonuclease Genes.

Fungal mitochondrial genomes act as "reservoirs" for homing endonucleases. These enzymes with their DNA site-specific cleavage activities are attractive tools for genome editing, targeted mutagenesis and gene therapy applications. Herein, we present strategies where homing endonuclease open reading frames (HEases ORFs) are interrupted with group II intron sequences. The ultimate goal is to achieve in vivo expression of HEases that can be regulated by manipulating the splicing efficiency of the HEase ORF-embedded group II introns. That addition of exogenous magnesium chloride (MgCl2) appears to stimulate splicing of nonnative group II introns in Escherichia coli and the addition of cobalt chloride (CoCl2) to the growth medium antagonizes the expression of HEase activity (i.e., splicing). Group II introns are potentially autocatalytic self-splicing elements and thus can be used as molecular switches that allow for temporal regulated HEase expression. This should be useful in precision genome engineering, mutagenesis, and minimizing off-target activities.

Using Group II Introns for Attenuating the In Vitro and In Vivo Expression of a Homing Endonuclease.

In Chaetomium thermophilum (DSM 1495) within the mitochondrial DNA (mtDNA) small ribosomal subunit (rns) gene a group IIA1 intron interrupts an open reading frame (ORF) encoded within a group I intron (mS1247). This arrangement offers the opportunity to examine if the nested group II intron could be utilized as a regulatory element for the expression of the homing endonuclease (HEase). Constructs were generated where the codon-optimized ORF was interrupted with either the native group IIA1 intron or a group IIB type intron. This study showed that the expression of the HEase (in vivo) in Escherichia coli can be regulated by manipulating the splicing efficiency of the HEase ORF-embedded group II introns. Exogenous magnesium chloride (MgCl2) stimulated the expression of a functional HEase but the addition of cobalt chloride (CoCl2) to growth media antagonized the expression of HEase activity. Ultimately the ability to attenuate HEase activity might be useful in precision genome engineering, minimizing off target activities, or where pathways have to be altered during a specific growth phase.

I-OmiI and I-OmiII: two intron-encoded homing endonucleases within the Ophiostoma minus rns gene.

The mitochondrial small subunit ribosomal RNA (rns) gene of the ascomycetous fungus Ophiostoma minus [strain WIN(M)371] was found to contain a group IC2 and a group IIB1 intron at positions mS569 and mS952 respectively. Both introns have open reading frames (ORFs) embedded that encode double motif LAGLIDADG homing endonucleases (I-OmiI and I-OmiII respectively). Codon-optimized versions of I-OmiI and I-OmiII were synthesized for overexpression in Escherichia coli. The in vitro characterization of I-OmiII showed that it is a functional homing endonuclease that cleaves the rns target site two nucleotides upstream (sense strand) of the intron insertion site generating 4 nucleotide 3' overhangs. The endonuclease activity of I-OmiII was tested using linear and circular substrates and cleavage activity was evaluated at various temperatures. The I-OmiI protein was expressed in E. coli, but purification was difficult, thus the endonuclease activity of this protein was tested via in vivo assays. Overall this study showed that there are many native forms of functional homing endonucleases yet to be discovered among fungal mtDNA genomes.

PCR-based bioprospecting for homing endonucleases in fungal mitochondrial rRNA genes.

Fungal mitochondrial genomes act as "reservoirs" for homing endonucleases. These enzymes with their DNA site-specific cleavage activities are attractive tools for genome editing and gene therapy applications. Bioprospecting and characterization of naturally occurring homing endonucleases offers an alternative to synthesizing artificial endonucleases. Here, we describe methods for PCR-based screening of fungal mitochondrial rRNA genes for homing endonuclease encoding sequences, and we also provide protocols for the purification and biochemical characterization of putative native homing endonucleases.

A homing endonuclease with a switch: characterization of a twintron encoded homing endonuclease.

The small ribosomal subunit gene residing in the mitochondrial DNA of the thermophilic fungus Chaetomium thermophilum var. thermophilum La Touche DSM 1495 is interrupted by a twintron at position mS1247. The mS1247 twintron represents the first mixed twintron found in fungal mtDNA, composed of an external group I intron encoding a LAGLIDADG open reading frame that is interrupted by an internal group II intron. Splicing of the internal group II intron reconstitutes the open reading frame and thus facilitates the expression of the encoded homing endonuclease. The cleavage assays suggest that the twintron encodes an active homing endonuclease that could potentially mobilize the twintron to rns genes that have not yet been invaded by this mobile composite element.