Precise genome editing across kingdoms of life using retron-derived DNA.

Exogenous DNA can be a template to precisely edit a cell's genome. However, the delivery of in vitro-produced DNA to target cells can be inefficient, and low abundance of template DNA may underlie the low rate of precise editing. One potential tool to produce template DNA inside cells is a retron, a bacterial retroelement involved in phage defense. However, little effort has been directed at optimizing retrons to produce designed sequences. Here, we identify modifications to the retron non-coding RNA (ncRNA) that result in more abundant reverse-transcribed DNA (RT-DNA). By testing architectures of the retron operon that enable efficient reverse transcription, we find that gains in DNA production are portable from prokaryotic to eukaryotic cells and result in more efficient genome editing. Finally, we show that retron RT-DNA can be used to precisely edit cultured human cells. These experiments provide a general framework to produce DNA using retrons for genome modification.

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

The bacterial retron reverse transcriptase system has served as an intracellular factory for single-stranded DNA in many biotechnological applications. In these technologies, a natural retron non-coding RNA (ncRNA) is modified to encode a template for the production of custom DNA sequences by reverse transcription. The efficiency of reverse transcription is a major limiting step for retron technologies, but we lack systematic knowledge of how to improve or maintain reverse transcription efficiency while changing the retron sequence for custom DNA production. Here, we test thousands of different modifications to the retron-Eco1 ncRNA and measure DNA production in pooled variant library experiments, identifying regions of the ncRNA that are tolerant and intolerant to modification. We apply this new information to a specific application: the use of the retron to produce a precise genome editing donor in combination with a CRISPR-Cas9 RNA-guided nuclease (an editron). We use high-throughput libraries in S. cerevisiae to additionally define design rules for editrons. We extend our new knowledge of retron DNA production and editron design rules to human genome editing to achieve the highest efficiency retron-Eco1 editrons to date.

Continuous Multiplexed Phage Genome Editing Using Recombitrons.

Bacteriophages, which naturally shape bacterial communities, can be co-opted as a biological technology to help eliminate pathogenic bacteria from our bodies and food supply1. 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 genomes2. 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 retrons3 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.