Optimization of in vivo activity of a bifunctional homing endonuclease and maturase reverses evolutionary degradation

Targeted manipulation of complex genomes often requires the introduction of a double-strand break at defined locations by site-specific DNA endonucleases. Here, we describe a monomeric nuclease domain derived from GIY-YIG homing endonucleases for genomeediting applications. Fusion of the GIY-YIG nuclease domain to threemember zinc-finger DNA binding domains generated chimeric GIYzinc finger endonucleases (GIY-ZFEs). Significantly, the I-TevI-derived fusions (Tev-ZFEs) function in vitro as monomers to introduce a double-strand break, and discriminate in vitro and in bacterial and yeast assays against substrates lacking a preferred 5′-CNNNG-3′ cleavage motif. The Tev-ZFEs function to induce recombination in a yeastbased assay with activity on par with a homodimeric Zif268 zincfinger nuclease. We also fused the I-TevI nuclease domain to a catalytically inactive LADGLIDADG homing endonuclease (LHE) scaffold. The monomeric Tev-LHEs are active in vivo and similarly discriminate against substrates lacking the 5′-CNNNG-3′ motif. The monomeric Tev-ZFEs and Tev-LHEs are distinct from the FokI-derived zinc-finger nuclease and TAL effector nuclease platforms as the GIY-YIG domain alleviates the requirement to design two nuclease fusions to target a given sequence, highlighting the diversity of nuclease domains with distinctive biochemical properties suitable for genome-editing applications.

Section: Discussionmentioning

confidence: 99%

Monomeric site-specific nucleases for genome editing

Kleinstiver

Wolfs

Kolaczyk

et al. 2012

“…For those enzymes that have accumulated mutations that reduce their stability or activity, random mutagenesis might be employed to rescue compromised function, as illustrated by prior studies of the WT I-AniI and DmoCre hybrid enzyme (10,41). Because the endonucleases within the I-OnuI subfamily appear to have recently diverged during their search for and invasion of a variety of "homing" sites, it is possible that many of them have retained most of their key residues for DNA binding and enzymatic activity.…”

Section: Discussionmentioning

confidence: 99%

“…Mutagenesis and selection of I-OnuI variants that cleave and disrupt the human MAO-B gene were conducted using protocols for selection described in ref. 41.…”

Section: Methodsmentioning

confidence: 99%

Tapping natural reservoirs of homing endonucleases for targeted gene modification

Takeuchi

Lambert²,

Mak

et al. 2011

Self Cite

110

Homing endonucleases mobilize their own genes by generating double-strand breaks at individual target sites within potential host DNA. Because of their high specificity, these proteins are used for “genome editing” in higher eukaryotes. However, alteration of homing endonuclease specificity is quite challenging. Here we describe the identification and phylogenetic analysis of over 200 naturally occurring LAGLIDADG homing endonucleases (LHEs). Biochemical and structural characterization of endonucleases from one clade within the phylogenetic tree demonstrates strong conservation of protein structure contrasted against highly diverged DNA target sites and indicates that a significant fraction of these proteins are sufficiently stable and active to serve as engineering scaffolds. This information was exploited to create a targeting enzyme to disrupt the endogenous monoamine oxidase B gene in human cells. The ubiquitous presence and diversity of LHEs described in this study may facilitate the creation of many tailored nucleases for genome editing.

“…Similarly, random mutagenesis screens for increased activity of I-CreI derivatives identified the A28 position in dimeric and monomeric I-CreI derivatives (G19 of I-CreI) (17,31). Likewise, up-activity mutants of I-AniI included randomly selected E-to-D substitutions in the equivalent position to E184 (32). In all cases, the coevolutionary context of the residues involved was not appreciated.…”

Section: Suppressor Mutations In the Coevolving Network Can Rescue Sumentioning

confidence: 99%

Control of catalytic efficiency by a coevolving network of catalytic and noncatalytic residues

McMurrough

Dickson

Thibert

et al. 2014

The active sites of enzymes consist of residues necessary for catalysis and structurally important noncatalytic residues that together maintain the architecture and function of the active site. Examples of evolutionary interactions between catalytic and noncatalytic residues have been difficult to define and experimentally validate due to a general intolerance of these residues to substitution. Here, using computational methods to predict coevolving residues, we identify a network of positions consisting of two catalytic metal-binding residues and two adjacent noncatalytic residues in LAGLIDADG homing endonucleases (LHEs). Distinct combinations of the four residues in the network map to distinct LHE subfamilies, with a striking distribution of the metal-binding Asp (D) and Glu (E) residues. Mutation of these four positions in three LHEs-I-LtrI, I-OnuI, and I-HjeMI-indicate that the combinations of residues tolerated are specific to each enzyme. Kinetic analyses under single-turnover conditions revealed that I-LtrI activity could be modulated over an ∼100-fold range by mutation of residues in the coevolving network. I-LtrI catalytic site variants with low activity could be rescued by compensatory mutations at adjacent noncatalytic sites that restore an optimal coevolving network and vice versa. Our results demonstrate that LHE activity is constrained by an evolutionary barrier of residues with strong context-dependent effects. Creation of optimal coevolving active-site networks is therefore an important consideration in engineering of LHEs and other enzymes.amino acid coevolution | genetic selection T he active sites of enzymes are often the most conserved positions in a multiple sequence alignment, as purifying selection for maintenance of function constrains amino acid variation of residues that directly participate in catalysis. Noncatalytic residues, often in close proximity to catalytic residues, contribute to enzymatic function by maintaining the architecture and chemical environment of the active site. Noncatalytic residues often show sequence variation in multiple sequence alignments, yet nonpermissive substitutions at these positions will have an impact on enzymatic function by disrupting the architecture or chemical environment necessary for catalysis. Thus, catalytic and noncatalytic residues must coevolve with each other and surrounding residues to maintain active-site conformation and chemistry and to buffer against potentially deleterious mutations (1, 2). Coevolving residues within a protein family can be predicted by computational methods that use mutual information theory to identify residue covariation in a multiple sequence alignment (2-5). However, because the magnitude of covariation between positions varies with the magnitude of positional variation (4), the identification of catalytic residues as part of coevolving networks is problematic for the simple reason that catalytic residues show little sequence variation. Although others have identified putative coevolution between residues involv...