Recording gene expression order in DNA by CRISPR addition of retron barcodes.

Biological processes depend on the differential expression of genes over time, but methods to make physical recordings of these processes are limited. Here we report a molecular system for making time-ordered recordings of transcriptional events into living genomes. We do this through engineered RNA barcodes, based on prokaryotic retrons1, that are reverse transcribed into DNA and integrated into the genome using the CRISPR-Cas system2. The unidirectional integration of barcodes by CRISPR integrases enables reconstruction of transcriptional event timing based on a physical record through simple, logical rules rather than relying on pretrained classifiers or post hoc inferential methods. For disambiguation in the field, we will refer to this system as a Retro-Cascorder.

Simultaneous multi-site editing of individual genomes using retron arrays.

During recent years, the use of libraries-scale genomic manipulations scaffolded on CRISPR guide RNAs have been transformative. However, these existing approaches are typically multiplexed across genomes. Unfortunately, building cells with multiple, nonadjacent precise mutations remains a laborious cycle of editing, isolating an edited cell and editing again. The use of bacterial retrons can overcome this limitation. Retrons are genetic systems composed of a reverse transcriptase and a noncoding RNA that contains an multicopy single-stranded DNA, which is reverse transcribed to produce multiple copies of single-stranded DNA. Here we describe a technology-termed a multitron-for precisely modifying multiple sites on a single genome simultaneously using retron arrays, in which multiple donor-encoding DNAs are produced from a single transcript. The multitron architecture is compatible with both recombineering in prokaryotic cells and CRISPR editing in eukaryotic cells. We demonstrate applications for this approach in molecular recording, genetic element minimization and metabolic engineering.

Retron reverse transcriptase termination and phage defense are dependent on host RNase H1.

Retrons are bacterial retroelements that produce single-stranded, reverse-transcribed DNA (RT-DNA) that is a critical part of a newly discovered phage defense system. Short retron RT-DNAs are produced from larger, structured RNAs via a unique 2'-5' initiation and a mechanism for precise termination that is not yet understood. Interestingly, retron reverse transcriptases (RTs) typically lack an RNase H domain and, therefore, depend on endogenous RNase H1 to remove RNA templates from RT-DNA. We find evidence for an expanded role of RNase H1 in the mechanism of RT-DNA termination, beyond the mere removal of RNA from RT-DNA:RNA hybrids. We show that endogenous RNase H1 determines the termination point of the retron RT-DNA, with differing effects across retron subtypes, and that these effects can be recapitulated using a reduced, in vitro system. We exclude mechanisms of termination that rely on steric effects of RNase H1 or RNA secondary structure and, instead, propose a model in which the tertiary structure of the single-stranded RT-DNA and remaining RNA template results in termination. Finally, we show that this mechanism affects cellular function, as retron-based phage defense is weaker in the absence of RNase H1.

Simultaneous multi-site editing of individual genomes using retron arrays.

Our understanding of genomics is limited by the scale of our genomic technologies. While libraries of genomic manipulations scaffolded on CRISPR gRNAs have been transformative, these existing approaches are typically multiplexed across genomes. Yet much of the complexity of real genomes is encoded within a genome across sites. Unfortunately, building cells with multiple, non-adjacent precise mutations remains a laborious cycle of editing, isolating an edited cell, and editing again. Here, we describe a technology for precisely modifying multiple sites on a single genome simultaneously. This technology - termed a multitron - is built from a heavily modified retron, in which multiple donor-encoding msds are produced from a single transcript. The multitron architecture is compatible with both recombineering in prokaryotic cells and CRISPR editing in eukaryotic cells. We demonstrate applications for this approach in molecular recording, genetic element minimization, and metabolic engineering.

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.