Precise Cas9 targeting enables genomic mutation prevention

Chavez, Alejandro; Pruitt, Benjamin W.; Tuttle, Marcelle; Shapiro, Rebecca; Cecchi, Ryan J; Winston, Jordan; Turczyk, Brian M.; Tung, Michael; Collins, James J.; Church, George M.

doi:10.1101/058974

Cited by 10 publications

(9 citation statements)

References 48 publications

(58 reference statements)

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“…14c). We think that the likely explanation why two of the tested 4T1-GLUT1 -/- tumors displayed elevated GLUT1 levels is due to the selection pressure on the tumor, which could revert a single mutation in one allele 36. Overall, these results demonstrate that the BiGluc technique performs very similarly to the gold standard technique in the field of in vivo glucose uptake imaging such as 18 F-FDG/PET.…”

Section: Resultsmentioning

confidence: 63%

Bioluminescent-based imaging and quantification of glucose uptake in vivo

et al. 2019

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Glucose is a major source of energy for most living organisms and its aberrant uptake is linked to many pathological conditions. However, our understanding of disease-associated glucose flux is limited due to the lack of robust tools. To date, positron emission tomography (PET) imaging remains the gold standard for measuring glucose uptake, and no optical tools exist for non-invasive longitudinal imaging of this important metabolite in in vivo settings. Here we report the development of a novel bioluminescent glucose uptake probe (BiGluc) for real-time, non-invasive longitudinal imaging of glucose absorption both in vitro and in vivo. In addition, we demonstrate that the sensitivity of our method is comparable with commonly used 18F-FDG-PET tracers and validate BiGluc as a tool for the identification of novel glucose transport inhibitors. The new imaging reagent enables a wide range of applications in the field of metabolism and drug development.

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Section: Resultsmentioning

confidence: 63%

Bioluminescent-based imaging and quantification of glucose uptake in vivo

et al. 2019

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“…2E). This stands in contrast to CRISPR-Cas methods in which hundreds of bases of homology are typically used (35,36). Notably, while efficiency exceeded 50% for all lengths attempted, donors of similar length can behave quite differently (Fig.…”

Section: Resultsmentioning

confidence: 85%

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

Schubert

Goodman

Wannier

et al. 2021

Proc. Natl. Acad. Sci. U.S.A.

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Creating and characterizing individual genetic variants remains limited in scale, compared to the tremendous variation both existing in nature and envisioned by genome engineers. Here we introduce retron library recombineering (RLR), a methodology for high-throughput functional screens that surpasses the scale and specificity of CRISPR-Cas methods. We use the targeted reverse-transcription activity of retrons to produce single-stranded DNA (ssDNA) in vivo, incorporating edits at >90% efficiency and enabling multiplexed applications. RLR simultaneously introduces many genomic variants, producing pooled and barcoded variant libraries addressable by targeted deep sequencing. We use RLR for pooled phenotyping of synthesized antibiotic resistance alleles, demonstrating quantitative measurement of relative growth rates. We also perform RLR using the sheared genomic DNA of an evolved bacterium, experimentally querying millions of sequences for causal variants, demonstrating that RLR is uniquely suited to utilize large pools of natural variation. Using ssDNA produced in vivo for pooled experiments presents avenues for exploring variation across the genome.

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“…Instead, many new extra-circuit mutations increased yEGFP::ZeoR levels, thereby improving drug resistance, without restoring broken PF function, indicating that evolution adopts alternate paths if they are available [50,51]. Restricting such alternate paths (e.g., by using higher drug concentrations or by preventing extra-circuit genomic mutations [52]) may facilitate functional reversions in future experiments. Importantly, some extra-circuit mutations did re-enable PF gene circuit function by elevating rtTA expression, indicating that evolutionary reversion is possible without any new mutations in rtTA or even the gene circuit.…”

Section: Discussionmentioning

confidence: 99%

Evolutionary regain of lost gene circuit function

Gouda

Manhart

Balazsi³

2019

Preprint

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Evolutionary reversibility -the ability to regain a lost function -is an important problem both in evolutionary and synthetic biology, where repairing natural or synthetic systems broken by evolutionary processes may be valuable. Here, we use a synthetic positivefeedback (PF) gene circuit integrated into haploid Saccharomyces cerevisiae cells to test if the population can restore lost PF function. In previous evolution experiments, mutations in a gene eliminated the fitness costs of PF activation. Since PF activation also provides drug resistance, exposing such compromised or broken mutants to both drug and inducer should create selection pressure to regain drug resistance and possibly PF function. Indeed, evolving seven PF mutant strains in the presence of drug revealed three adaptation scenarios through genomic mutations outside of the PF circuit that elevate PF basal expression, possibly by affecting transcription, translation, degradation and other fundamental cell functions. Nonfunctional mutants gained drug resistance without ever developing high expression, while quasi-functional and dysfunctional PF mutants developed high expression which then diminished, although more slowly for dysfunctional mutants where revertant clones arose. These results highlight how intracellular context, such as the growth rate, can affect regulatory network dynamics and evolutionary dynamics, which has important consequences for understanding the evolution of drug resistance and developing future synthetic biology applications. Significance Statement Natural or synthetic genetic modules can lose their function over long-term evolution if the function is costly. How populations can evolve to restore broken functions is poorly understood. To test the reversibility of evolutionary breakdown, we use yeast cell populations with a chromosomally integrated synthetic gene circuit. In previous evolution experiments the gene circuit lost its costly function through various mutations. By exposing such mutant populations to conditions where regaining gene circuit function would be beneficial we find adaptation scenarios with or without repairing lost gene circuit function. These results are important for drug resistance or future synthetic biology applications where loss and regain of function play a significant role.

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Precise Cas9 targeting enables genomic mutation prevention

Cited by 10 publications

References 48 publications

Bioluminescent-based imaging and quantification of glucose uptake in vivo

Bioluminescent-based imaging and quantification of glucose uptake in vivo

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

Evolutionary regain of lost gene circuit function

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