Large-scale discovery of recombinases for integrating DNA into the human genome

Abstract

SUMMARYRecent microbial genome sequencing efforts have revealed a vast reservoir of mobile genetic elements containing integrases that could be useful genome engineering tools. Large serine recombinases (LSRs), such as Bxb1 and PhiC31, are bacteriophage-encoded integrases that can facilitate the insertion of phage DNA into bacterial genomes. However, only a few LSRs have been previously characterized and they have limited efficiency in human cells. Here, we developed a systematic computational discovery workflow that identifies thousands of new LSRs and their cognate DNA attachment sites by. We validate this approach via experimental characterization of LSRs in human cells, leading to three classes of LSRs distinguished from one another by their efficiency and specificity. We identify landing pad LSRs that efficiently integrate into synthetically installed attachment sites orthogonal to the human genome, human genome-targeting LSRs with computationally predictable pseudosites, and multi-targeting LSRs that can unidirectionally integrate cargos at with similar efficiency and superior specificity to commonly used transposases. LSRs from each category were functionally characterized in human cells, overall achieving up to 7-fold higher plasmid recombination than Bxb1 and genome insertion efficiencies of 40-70% with cargo sizes over 7 kb. Overall, we establish a paradigm for large-scale discovery of microbial recombinases and reconstruction of their target sites directly from microbial sequencing data. This strategy provides a rich resource of over 60 experimentally characterized LSRs that can function in human cells and thousands of additional candidates for large-payload genome editing without exposed DNA double-stranded breaks.

Instantaneous visual genotyping and facile site-specific transgenesis via CRISPR-Cas9 and phiC31 integrase

“…Thus, this will be a useful future direction for zebrafish. Moreover, it will be helpful to identify other microbial DNA integrases that exhibit high efficiencies in zebrafish, which will enable more sophisticated experimental designs involving multiple DNA integrations or cassette exchange (Durrant, 2021; Jusiak et al, 2019; Low et al, 2022). It can be expected that a broader adoption and more creative uses of the CRISPR and integrase technologies in zebrafish and other model organisms will play an important role in accelerating more transformative biomedical research in the near future.…”

Mobile and Self‐Sustained Data Storage in an Extremophile Genomic DNA

“…Among the integrases that have been shown to work in E. coli, very few had been proven functional in a different species. [47][48][49][50][51] Through a comprehensive screen, we obtained 16 integrases that successfully worked in H. bluephagenesis (Table S1, Supporting Information), with each recognizing a specific pair of attB and attP sites with extraordinary sequence specificity.…”

Genomically mined acoustic reporter genes enable real-timein vivomonitoring of tumors and tumor-homing probiotics

“…These qualities would enable longterm noninvasive imaging of gene expression in a broad range of in vivo applications. We hypothesized that 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] -would yield ARGs with improved properties, which could be further optimized through genetic engineering. By cloning and screening 15 distinct polycistronic operons from a diverse set of GV-expressing species representing a broad phylogeny, we identified two GV gene clusters -from Serratia sp.…”