Expanding the genome editing toolbox by rational reprogramming of tyrosine recombinase DNA specificities

Introduction

Gene therapies aim to cure genetic diseases by modifying the DNA blueprint. An ideal gene therapy should be durable, safe, and efficient, which often requires integration of large therapeutic DNA cargo at precise genomic locations.

Limitations of Current Technologies

Advanced DNA editors with programmable specificities, such as the CRISPR technology, are limited by the nuclease-based mechanism and poor efficiency of large DNA insertions. Increased efficiency, specificity, and precision of large edits are required to address a broad therapeutic space.

Tyrosine Recombinases

Tyrosine recombinases (YRs) efficiently rearrange large DNA segments without the pitfalls of nuclease-based editors, but with an important caveat – rational reprogramming of their specificities for binding non-native DNA targets is an unsolved problem.

Project Goals

EditYR ambitiously aims to develop a rationally programmable tyrosine recombinase platform for efficient integration of large DNA cargo into precise genomic locations. EditYR will expand recombinase engineering capabilities by exploiting the recent advances in large scale DNA synthesis and sequencing coupled with bioinformatics and innovative protein design.

Objectives

1. Objective 1: Engineer tyrosine recombinase DNA specificities and extract a comprehensive DNA recognition code.
2. Objective 2: Modify their oligomerization properties to enable binding non-palindromic targets, allowing targeting of any desired nucleotide sequence.
3. Objective 3: Investigate requirements for irreversible recombination to maximize integration efficiencies and unlock the full therapeutic potential of these enzymes.
4. Objective 4: Validate engineered enzymes by identification of targetable sequences in therapeutically relevant genomic regions and integration of therapeutic DNA cargo in various human cell types for different disease cases.

Conclusion

EditYR will establish tyrosine recombinases as advanced tools for genome editing, creating a DNA editing technology that will elevate the capabilities of gene therapies to unprecedented levels.