Systematic discovery of recombinases for efficient integration of large DNA sequences into the human genome

Abstract

Large serine recombinases (LSRs) are DNA integrases that facilitate the site-specific integration of mobile genetic elements into bacterial genomes. Only a few LSRs, such as Bxb1 and PhiC31, have been characterized to date, with limited efficiency as tools for DNA integration in human cells. In this study, we developed a computational approach to identify thousands of LSRs and their DNA attachment sites, expanding known LSR diversity by >100-fold and enabling the prediction of their insertion site specificities. We tested their recombination activity in human cells, classifying them as landing pad, genome-targeting or multi-targeting LSRs. Overall, we achieved up to seven-fold higher recombination than Bxb1 and genome integration efficiencies of 40–75% with cargo sizes over 7 kb. We also demonstrate virus-free, direct integration of plasmid or amplicon libraries for improved functional genomics applications. This systematic discovery of recombinases directly from microbial sequencing data provides a resource of over 60 LSRs experimentally characterized in human cells for large-payload genome insertion without exposed DNA double-stranded breaks.

High-throughput genetic engineering of nonmodel and undomesticated bacteria via iterative site-specific genome integration

“…Some of the recombinases function at high efficiency and high accuracy, but others are more variable between organisms. Recently, many new recombinases and corresponding att sites have been computationally predicted ( 70 , 71 ). These recombinases will serve as a rich source of new, diverse enzymes for the extension of SAGE-based approaches.…”

Genomically mined acoustic reporter genes for real-time in vivo monitoring of tumors and tumor-homing bacteria

“…We sought to make acoustic proteins widely useful in in vivo biological research and potential clinical applications by developing ARGs that, when expressed heterologously in either bacteria or mammalian cancer cell lines, could produce GVs with strong non-linear ultrasound contrast and enable long-term expression under physiological conditions. We used a genomic mining approach-previously applied to improving fluorescent proteins [11][12][13][14] , opsins [15][16][17] , Cas proteins [18][19][20][21][22] and other biotechnology tools [23][24][25][26][27][28] -to identify ARGs with improved properties, which we subsequently optimized through genetic engineering. By cloning and screening 15 distinct polycistronic operons chosen from a diverse set of 288 GV-expressing species representing a broad phylogeny, we identified two GV gene clusters-from Serratia sp.…”

High fidelity one‐pot DNA assembly using orthogonal serine integrases

“…Identification and characterization of more LSI RDFs will also enhance the flexibility of these systems for use in precision and programmable DNA rearrangements. [8,9,22,26,61,62] AUTHOR CONTRIBUTIONS…”

“…The availability of a large palette of orthogonal LSIs is becoming more realistic as more of these enzymes are being isolated and characterized using computational genome mining tools. [6,61,62]…”