Pairing of single mutations yields obligate Cre-type site-specific recombinases

Abstract

Tyrosine site-specific recombinases (SSRs) represent a versatile genome editing tool with considerable therapeutic potential. Recent developments to engineer and evolve SSRs into heterotetramers to improve target site flexibility signified a critical step towards their broad utility in genome editing. However, SSR monomers can form combinations of different homo- and heterotetramers in cells, increasing their off-target potential. Here, we discover that two paired mutations targeting residues implicated in catalysis lead to simple obligate tyrosine SSR systems, where the presence of all distinct subunits to bind as a heterotetramer is obligatory for catalysis. Therefore, only when the paired mutations are applied as single mutations on each recombinase subunit, the engineered SSRs can efficiently recombine the intended target sequence, while the subunits carrying the point mutations expressed in isolation are inactive. We demonstrate the utility of the obligate SSR system to improve recombination specificity of a designer-recombinase for a therapeutic target in human cells. Furthermore, we show that the mutations render the naturally occurring SSRs, Cre and Vika, obligately heteromeric for catalytic proficiency, providing a straight-forward approach to improve their applied properties. These results facilitate the development of safe and effective therapeutic designer-recombinases and advance our mechanistic understanding of SSR catalysis.

Prediction of designer-recombinases for DNA editing with generative deep learning

“…So far, the development of designer-recombinases for novel target sites has been accomplished by applying directed molecular evolution [4][5][6][7]9,40,41,[44][45][46][47][48] , an effective but laborious and time consuming process. A more direct and faster approach could be the intelligent design of Y-SSRs for predefined target sequences.…”

“…This bottleneck represents a considerable hurdle to harness the full potential of designer-recombinases as a versatile genome editing tool.Current approaches to adapt site-specific recombinases to novel target sequences use directed molecular evolution. One powerful approach to evolve designer Y-SSRs utilizes a plasmid-based bacterial application called substrate-linked directed evolution [3][4][5][6][7]. SLiDE allows libraries of recombinases to be progressively evolved by screening random mutations in conjunction with a selection scheme for activity on new, stepwise altered target sequences.…”

Prediction of designer-recombinases for DNA editing with generative deep learning

“…So far, the development of designer-recombinases for novel target sites has been accomplished by applying directed molecular evolution (Buchholz et al 1998; Buchholz and Stewart 2001; Rufer 2002; Santoro and Schultz 2002; Voziyanov et al 2002; Voziyanov et al 2003; Bolusani et al 2006; Sarkar et al 2007; Shah et al 2015; Karpinski et al 2016; Voziyanova et al 2016; Lansing et al 2019; Hoersten et al 2021; Lansing et al 2022), an effective but laborious and time consuming process. A more direct and faster approach could be the intelligent design of Y-SSRs for predefined target sequences.…”

“…Current approaches to adapt site specific recombinases to novel target sequences use directed molecular evolution. One powerful approach to evolve designer Y-SSRs utilizes a plasmid-based bacterial application called substrate-linked directed evolution (SLiDE, (Buchholz and Stewart 2001; Buchholz and Hauber 2011; Lansing et al 2019; Hoersten et al 2021; Lansing et al 2022)). SLiDE allows libraries of recombinases to be progressively evolved by screening random mutations in conjunction with a selection scheme for activity on new, stepwise altered target sequences.…”

A Novel Cre/lox71-Based System for Inducible Expression of Recombinant Proteins and Genome Editing

“…The specificity of the developed induction system requires further investigation, although it is extremely specific according to the literature [ 33 , 34 ], which states that Cre-mediated recombination can be used for creating cellular and animal transgenic models [ 35 , 36 ]. We expect that recent, cautious reports on the possible off-target activity of Cre recombinase [ 37 ] will not be an obstacle to the usage of such a system for research or biotechnological purposes since this modification of the Cre/lox71 system is active mainly within the promoter and coding regions of the genome, and for significant activation of gene expression it requires the presence of several closely grouped transactivator binding sites.…”