Efficient, continuous mutagenesis in human cells using a pseudo-random DNA editor

Chen, Haiqi; Liu, Sophia; Padula, Samuel; Lesman, Daniel; Griswold, Kettner; Lin, Allen; Zhao, Tuanjie; Marshall, Jamie L.; Chen, Fei

doi:10.1038/s41587-019-0331-8

Cited by 71 publications

(93 citation statements)

References 27 publications

(33 reference statements)

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“…By placing the P T7 at the 3′-end of the target gene, in reverse orientation, expression of the target gene can be preserved from its endogenous 5′-promoter recognized by the host RNAP. Two recent independent studies have also reported the mutagenic activity of CD fusions to T7RNAP, in E. coli 21 and in mammalian cells 22 . Moore et al 21 described a chimeric fusion between rAPOBEC1 and T7RNAP (MutaT7) that in E. coli introduces mutations within a plasmid DNA segment downstream of P T7 .…”

Section: Introductionmentioning

confidence: 93%

“…The study in mammalian cells uses fusions of rAPOBEC1 or a hyperactive mutant AID to T7RNAP. Mutations in a DNA segment of at least 2000 bp were reported, but no brake system was included to delimit the edited window 22 . In this work, we describe the mutagenic action of different BDs (i.e., AID, rAPOBEC1, pmCDA1, and TadA*) fused to T7RNAP on a target genomic DNA segment in E. coli chromosome.…”

Section: Introductionmentioning

confidence: 99%

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In vivo diversification of target genomic sites using processive base deaminase fusions blocked by dCas9

et al. 2020

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In vivo mutagenesis systems accelerate directed protein evolution but often show restricted capabilities and deleterious off-site mutations on cells. To overcome these limitations, here we report an in vivo platform to diversify specific DNA segments based on protein fusions between various base deaminases (BD) and the T7 RNA polymerase (T7RNAP) that recognizes a cognate promoter oriented towards the target sequence. Transcriptional elongation of these fusions generates transitions C to T or A to G on both DNA strands and in long DNA segments. To delimit the boundaries of the diversified DNA, the catalytically dead Cas9 (dCas9) is tethered with custom-designed crRNAs as a “roadblock” for BD-T7RNAP elongation. Using this T7-targeted dCas9-limited in vivo mutagenesis (T7-DIVA) system, rapid molecular evolution of the antibiotic resistance gene TEM-1 is achieved. While the efficiency is demonstrated in E. coli, the system can be adapted to a variety of bacterial and eukaryotic hosts.

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Section: Introductionmentioning

confidence: 93%

Section: Introductionmentioning

confidence: 99%

In vivo diversification of target genomic sites using processive base deaminase fusions blocked by dCas9

et al. 2020

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“…Although indels are applicable to regulatory regions and even coding sequences [55][56][57] , point mutagenesis would enable fi ne mapping of regulatory nucleotides and amino acids. This exciting possibility could be opened up by implementing hyperactive base [58][59][60][61] -or prime editors [62][63][64] . Alternatives to CRISPR-Cas could be developed based on inducible expression of cassette exchange 65,66 or targeted transposons 67,68 .…”

Section: Discussionmentioning

confidence: 99%

Parallel genetics of regulatory sequencesin vivo

Froehlich

Uyar

Herzog

et al. 2020

Preprint

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Understanding how regulatory sequences control gene expression is fundamental to explain how phenotypes arise in health and disease. Traditional reporter assays inform about function of individual regulatory elements, typically in isolation. However, regulatory elements must ultimately be understood by perturbing them within their genomic environment and developmental- or tissue-specific contexts. This is technically challenging; therefore, few regulatory elements have been characterized in vivo. Here, we used inducible Cas9 and multiplexed guide RNAs to create hundreds of mutations in enhancers/promoters and 3′ UTRs of 16 genes in C. elegans. To quantify the consequences of mutations on expression, we developed a targeted RNA sequencing strategy across hundreds of mutant animals. We were also able to systematically and quantitatively assign fitness cost to mutations. Finally, we identified and characterized sequence elements that strongly regulate phenotypic traits. Our approach enables highly parallelized, functional analysis of regulatory sequences in vivo.

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“…Both of those limitations inherent to exploring long time scales of adaptation using barcoding approaches would likely be mitigated by using an approach that allows either periodic introduction of additional barcodes (as in 38 ), or allows modification of barcodes over time (e.g. 9 ), to maintain barcode diversity within the evolving populations, and measuring fitness of isolated clones in each of the environments at each environmental switch.…”

Section: Discussionmentioning

confidence: 99%

Adaptation is influenced by the complexity of environmental change during evolution in a dynamic environment

Boyer

Sherlock

2019

Preprint

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The environmental conditions of microorganisms' habitats may fluctuate in unpredictable ways, in terms of temperature, carbon source, pH, and salinity to name but a few. Such environmental heterogeneity presents a challenge for such microorganisms, as they have to adapt not only to be fit under a specific condition, but they must also be robust across many conditions and be able to deal with the switch between conditions itself. While experimental evolution has been used for decades to gain insight into the adaptive process, this has been largely in unvarying conditions. In cases where changing environments have been investigated, relatively little is known about how such environment influence the dynamics of the adaptive process itself, as well as the genetic and phenotypic outcomes. We designed a systematic series of evolution experiments where we used two conditions with differing time-scales of adaptation and varied the rate of switching between them. We used lineage tracking to follow adaption itself, and whole genome sequenced adaptive clones from each of the experiments. We find that the both the switch rate and the order of the conditions influences adaptation, and that switching can both speed up and slow down adaptation, depending on those parameters. We also find different adaptive outcomes, both at the genetic and phenotypic level, even if populations spent the same amount of total time in the two different conditions, but that the order and/or switch rate differed. Thus, in a variable environment adaptation depends not only on the nature of the conditions and phenotypes under selection, but also on the complexity of the manner in which those conditions are combined to result in a given dynamic environment.

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Efficient, continuous mutagenesis in human cells using a pseudo-random DNA editor

Cited by 71 publications

References 27 publications

In vivo diversification of target genomic sites using processive base deaminase fusions blocked by dCas9

In vivo diversification of target genomic sites using processive base deaminase fusions blocked by dCas9

Parallel genetics of regulatory sequencesin vivo

Adaptation is influenced by the complexity of environmental change during evolution in a dynamic environment

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