Retroelement-Based Genome Editing and Evolution

Simon, Anna J.; Morrow, Barrett R.; Ellington, Andrew D.

doi:10.1021/acssynbio.8b00273

Cited by 51 publications

(54 citation statements)

References 72 publications

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“…However, the sequences and lengths of the reverse transcribed template significantly vary and frequently show no sequence similarity between retrons 7 . The ability of retrons to produce ssDNA in situ has been adapted for multiple applications of synthetic biology and genome engineering 5,[8][9][10] .…”

Section: Introductionmentioning

confidence: 99%

Bacterial retrons function in anti-phage defense

Millman

Bernheim

Stokar-Avihail

et al. 2020

Preprint

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Retrons are bacterial genetic elements comprised of a reverse transcriptase (RT) and a non-coding RNA. The RT uses the non-coding RNA as a template, generating a chimeric RNA/DNA molecule in which the RNA and DNA components are covalently linked. Although retrons were discovered three decades ago, their function remained unknown. In this study we report that retrons function as anti-phage defense systems. The defensive unit is composed of three components: the RT, the non-coding RNA, and an effector protein. Retron-containing systems are abundant in genomic "defense islands", suggesting a role for most retrons in phage resistance. By cloning multiple retron systems into a retron-less Escherichia coli strain, we show that these systems confer defense against a broad range of phages, with different retrons defending against different phages. Focusing on a single retron, Ec48, we show evidence that it is a "guardian" of RecBCD, a complex with central anti-phage functions in the bacterial cell. Inhibition of RecBCD by dedicated phage proteins activates the retron, leading to abortive infection and cell death. Thus, the Ec48 retron forms a second line of defense that is triggered if the first lines of defense have collapsed. Our results expose a new family of anti-phage defense systems abundant in bacteria.

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Section: Introductionmentioning

confidence: 99%

Bacterial retrons function in anti-phage defense

Millman

Bernheim

Stokar-Avihail

et al. 2020

Preprint

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“…Additionally, previous studies have demonstrated that inactivation of exonucleases improves oligonucleotide recombineering by up to 3-fold 20,21 , and this effect has been shown for retron recombineering as well 22,23 . Inactivation of exonuclease genes recJ or sbcB (also known as xonA ) individually provided benefit, and inactivation of both together increased the fraction of genomes edited during induction of retron recombineering in batch growth by 131 and 201-fold for the gyrA and rpoB donors, respectively (Fig 2B, S1C).…”

Section: Resultsmentioning

confidence: 80%

High throughput functional variant screens via in-vivo production of single-stranded DNA

Schubert

Goodman

Wannier

et al. 2020

Preprint

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5Tremendous genetic variation exists in nature, but our ability to create and characterize 6 individual genetic variants remains far more limited in scale. Likewise, engineering proteins and 7 phenotypes requires the introduction of synthetic variants, but design of variants outpaces 8 experimental measurement of variant effect. Here, we optimize efficient and continuous 9 generation of precise genomic edits in Escherichia coli, via in-vivo production of single-stranded 10 DNA by the targeted reverse-transcription activity of retrons. Greater than 90% editing 11 efficiency can be obtained using this method, enabling multiplexed applications. We introduce 12 Retron Library Recombineering (RLR), a system for high-throughput screens of variants, 13 wherein the association of introduced edits with their retron elements enables a targeted deep 14 sequencing phenotypic output. We use RLR for pooled, quantitative phenotyping of synthesized 15 variants, characterizing antibiotic resistance alleles. We also perform RLR using sheared 16 genomic DNA of an evolved bacterium, experimentally querying millions of sequences for 17 antibiotic resistance variants. In doing so, we demonstrate that RLR is uniquely suited to utilize 18 non-designed sources of variation. Pooled experiments using ssDNA produced in vivo thus 19 present new avenues for exploring variation, both designed and not, across the entire genome. 20 21 Introduction: 22Constructing genotypes of interest and observing their effect on phenotype critically aids 23 our understanding of genetics and genome function. As methods for editing genomes have 24 progressed, this "reverse genetics" approach has expanded in breadth and scale, from knockout 25 libraries 1 to refactored genomes 2,3 . These experiments can now be performed within multiplexed 26 pools, which allow an ever greater number of mutations to be explored across varied conditions. 27Critically, both creating genotypes and observing phenotype within pools has necessitated 28 2 development of new techniques. Transposon insertions 4 , marked integrations 5 and CRISPR-29 inhibition 6,7 can create thousands of variants simultaneously within pooled experiments, and 30 targeted sequencing of these elements enables pooled measurement of variant phenotypes. These 31 advancements in experimental scale have fundamentally transformed our understanding of 32 genome function 8 . 33However, these current high-throughput genetic techniques remain limited, in that they 34 typically introduce, ablate, or regulate kilobases of DNA to create variation. This contrasts with 35 point mutations, which are ubiquitous in natural variation 9 , and are indispensable for engineering 36 proteins 10 and metabolic pathways 11 . While modifying kilobases of DNA can add and subtract 37 functional elements such as genes and regulatory sequences from the genome, point mutations 38 can alter the function of these elements, accessing a larger phenotypic landscape. 39Point mutations and other precision edits can be performed by oligonucleo...

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“…First, we varied the conditions that affect the efficiency of homologous recombination. The effects of changing the length of msd (from 55 to 115 bp) 18, 22 and the presence/absence of Beta recombinase 13, 14 (which stabilizes ssDNA) were tested; optimal efficiency was obtained with an msd length of 95 bp and co-expression of Beta recombinase ( Figure 2d, e ). We then examined the effect of varying conditions related to selection pressure using the CRISPR/Cas9 system.…”

Section: Resultsmentioning

confidence: 99%

“…Unedited cells can be problematic because their presence can interfere with interpretation when the screening process is not based on chemical selection. Enhancing the recombineering machinery via knock-out of exonucleases 14 , for example, could help reduce the proportion of unedited cells.…”

Section: Resultsmentioning

confidence: 99%

“…To overcome this challenge, a recently developed Cas9 retron precise parallel editing via homology (CRISPEY) 10 method uses the retron system 11, 12 for donor DNA expression instead of separate transfection of donor DNA fragments. The retron system for Escherichia coli 13, 14 , comprising msr, msd, and a reverse transcriptase (RT), was modified to express single-stranded donor DNAs in yeast, and the modified retron was flanked by sgRNA. CRISPEY thus enables the preparation of both sgRNA and donor DNA using a single vector.…”

Section: Introductionmentioning

confidence: 99%

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Multiplex generation, tracking, and functional screening of substitution mutants using a CRISPR/retron system

Lim

Jun

Park

et al. 2020

Preprint

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ABSTRACTWe developed a clustered regularly interspaced short palindromic repeats (CRISPR)/retron system for multiplexed generation of substitution mutations by co-utilization of a retron system that continuously expresses donor DNA and a CRISPR/Cas9 cassette that induces cleavage at target genomic loci. Our system efficiently introduces substitution mutation in the Escherichia coli genome in a high-throughput manner. These substitution mutations can be tracked by analysis of retron plasmid sequences without laborious amplification of individual edited loci. We demonstrated that our CRISPR/retron system can introduce thousands of mutations in a single experiment and be used for screening phenotypes related to chemical responses or fitness changes. We expect that our system could facilitate genome-scale substitution screenings.

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Retroelement-Based Genome Editing and Evolution

Cited by 51 publications

References 72 publications

Bacterial retrons function in anti-phage defense

Bacterial retrons function in anti-phage defense

High throughput functional variant screens via in-vivo production of single-stranded DNA

Multiplex generation, tracking, and functional screening of substitution mutants using a CRISPR/retron system

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