Engineering domain fusion chimeras from I-OnuI family LAGLIDADG homing endonucleases

Abstract

Although engineered LAGLIDADG homing endonucleases (LHEs) are finding increasing applications in biotechnology, their generation remains a challenging, industrial-scale process. As new single-chain LAGLIDADG nuclease scaffolds are identified, however, an alternative paradigm is emerging: identification of an LHE scaffold whose native cleavage site is a close match to a desired target sequence, followed by small-scale engineering to modestly refine recognition specificity. The application of this paradigm could be accelerated if methods were available for fusing N- and C-terminal domains from newly identified LHEs into chimeric enzymes with hybrid cleavage sites. Here we have analyzed the structural requirements for fusion of domains extracted from six single-chain I-OnuI family LHEs, spanning 40–70% amino acid identity. Our analyses demonstrate that both the LAGLIDADG helical interface residues and the linker peptide composition have important effects on the stability and activity of chimeric enzymes. Using a simple domain fusion method in which linker peptide residues predicted to contact their respective domains are retained, and in which limited variation is introduced into the LAGLIDADG helix and nearby interface residues, catalytically active enzymes were recoverable for ∼70% of domain chimeras. This method will be useful for creating large numbers of chimeric LHEs for genome engineering applications.

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

“…A common strategy for engineering LHEs with altered specificity is to fuse two halves (or domains) from different LHEs, each with different DNA-binding specificity (14,16,(35)(36)(37)(38). This strategy has been successful, particularly when the N-and C-terminal domains are derived from closely related LHEs.…”

“…Moreover, the monomeric and dimeric LHEs likely evolved under different functional constraints, and the phylogenetic signal and functional information for either form is diluted in alignments that include both the monomeric and dimeric LHEs. Because LHEs are currently under investigation for use as genome-editing agents (16)(17)(18), a greater understanding of their functional constraints would aid in engineering studies.…”

Engineering and Flow-Cytometric Analysis of Chimeric LAGLIDADG Homing Endonucleases from Homologous I-OnuI-Family Enzymes

“…In our experience, up to 50 % of I-OnuI family chimeras show significant cleavage activity with this direct domain fusion method [4]. However, certain enzyme combinations will be incompatible and require further engineering to achieve DNA cleavage activity.…”

“…The structural dissimilarity of these two parent enzymes made necessary both computational modeling and in vitro selection to repack the chimeric interface. Based on this work, it was hypothesized and confirmed that the generation of chimeras from within a family of highly homologous monomeric LHEs could produce a large number of active enzymes with minimal engineering [4]. …”

“…These N- and C-terminal halves are then recombined and joined with an artificial linker. In our experience, the choice of protein linker can have a significant effect on the stability and activity of the resulting chimera, so for the sake of simplicity and overall compatibility, we suggest using a “half-and-half” hybrid linker [4]. This strategy incorporates residues from both native parents to maintain important interactions with each respective domain, and introduces a flexible and minimally invasive tri-residue bridge at the point of fusion.…”