Continuous Multiplexed Phage Genome Editing Using Recombitrons

Abstract

Bacteriophages, which naturally shape bacterial communities, can be co-opted as a biological technology to help eliminate pathogenic bacteria from our bodies and food supply. Phage genome editing is a critical tool to engineer more effective phage technologies. However, editing phage genomes has traditionally been a low efficiency process that requires laborious screening, counter selection, or in vitro construction of modified genomes. These requirements impose limitations on the type and throughput of phage modifications, which in turn limit our knowledge and potential for innovation. Here, we present a scalable approach for engineering phage genomes using recombitrons: modified bacterial retrons that generate recombineering donor DNA paired with single stranded binding and annealing proteins to integrate those donors into phage genomes. This system can efficiently create genome modifications in multiple phages without the need for counterselection. Moreover, the process is continuous, with edits accumulating in the phage genome the longer the phage is cultured with the host, and multiplexable, with different editing hosts contributing distinct mutations along the genome of a phage in a mixed culture. In lambda phage, as an example, recombitrons yield single-base substitutions at up to 99% efficiency and up to 5 distinct mutations installed on a single phage genome, all without counterselection and only a few hours of hands-on time.

An experimental census of retrons for DNA production and genome editing

“…The ability of retrons to continuously produce a ssDNA containing a precise target mutation has been recently exploited for recombineering purposes in both bacteria and phages 13,16,18,[21][22][23] . Retron-based recombineering relies on the reverse transcription of a modified RT-DNA that contains an editing donor (Fig 3a).…”

High Throughput Variant Libraries and Machine Learning Yield Design Rules for Retron Gene Editors

“…In bacteria, retrons are a tripartite anti-phage system composed of a reverse transcriptase (RT), a non-coding RNA (ncRNA) that is reverse transcribed into DNA (RT-DNA), and an effector protein 1,2 . In biotechnology, the retron RT is used to reverse transcribe modified forms of retron ncRNA into RT-DNA that has been used as: donor DNA for precise editing in bacteria [3][4][5][6][7][8] , bacteriophage [9][10][11] , plants 12,13 , and eukaryotes 5,[14][15][16][17] ; DNA barcodes to record molecular events 18,19 ; DNA containing transcription factor motifs for transcription factor activity attenuation 20 ; DNA aptamers 21 ; and DNAzymes for mRNA cleavage 22 .…”