Redesigning Recombinase Specificity for Safe Harbor Sites in the Human Genome

Area of Science:

• Molecular Biology
• Genetic Engineering
• Protein Engineering

Background:

• Site-specific recombinases (SSRs) are crucial for precise DNA manipulation in genetic engineering.
• Engineered SSRs, like zinc-finger and TAL effector recombinases, offer customizable DNA recognition.
• Current engineered SSRs face limitations due to inherent catalytic domain base requirements.

Purpose of the Study:

• To expand the targeting repertoire of engineered recombinases.
• To overcome limitations in catalytic specificity of existing SSRs.
• To develop novel recombinases with broader DNA recognition for genome engineering.

Main Methods:

• Utilized a genetic screen to isolate enhanced mutants of Bin and Tn21 recombinases.
• Determined the specific base requirements for recombination for the isolated mutants.
• Selected for variants capable of recombining target sites in the human CCR5 gene and AAVS1 locus.

Main Results:

• Successfully isolated enhanced mutants of Bin and Tn21 recombinases.
• Identified specific base requirements enabling recognition of new target sites.
• Demonstrated genome engineering potential by targeting specific human gene loci.

Conclusions:

• Protein engineering combined with functional characterization effectively expands recombinase targeting capabilities.
• Developed novel recombinases with enhanced and expanded DNA targeting specificity.
• These findings pave the way for more versatile genome engineering tools.