A transient reporter for editing enrichment (TREE) in human cells

Standage-Beier, Kylie; Tekel, Stefan J.; Brookhouser, Nicholas; Schwarz, Grace; Nguyen, Toan; Wang, Xiao; Brafman, David A.

doi:10.1093/nar/gkz713

Cited by 39 publications

(22 citation statements)

References 59 publications

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“…First, it provides a universal solution to enrich for a variety of editing events in cells, including single-nucleotide polymorphisms, small insertions and deletions, and precise large-fragment knock-ins. These edits can be achieved without the need for an exogenous selection marker, unlike other selection methods involving Cas9 nuclease or base editors 13 , 15 , 16 .…”

Section: Discussionmentioning

confidence: 99%

Universal toxin-based selection for precise genome engineering in human cells

Akrap

Cerboni

et al. 2021

Nat Commun

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Prokaryotic restriction enzymes, recombinases and Cas proteins are powerful DNA engineering and genome editing tools. However, in many primary cell types, the efficiency of genome editing remains low, impeding the development of gene- and cell-based therapeutic applications. A safe strategy for robust and efficient enrichment of precisely genetically engineered cells is urgently required. Here, we screen for mutations in the receptor for Diphtheria Toxin (DT) which protect human cells from DT. Selection for cells with an edited DT receptor variant enriches for simultaneously introduced, precisely targeted gene modifications at a second independent locus, such as nucleotide substitutions and DNA insertions. Our method enables the rapid generation of a homogenous cell population with bi-allelic integration of a DNA cassette at the selection locus, without clonal isolation. Toxin-based selection works in both cancer-transformed and non-transformed cells, including human induced pluripotent stem cells and human primary T-lymphocytes, as well as it is applicable also in vivo, in mice with humanized liver. This work represents a flexible, precise, and efficient selection strategy to engineer cells using CRISPR-Cas and base editing systems.

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Section: Discussionmentioning

confidence: 99%

Universal toxin-based selection for precise genome engineering in human cells

Akrap

Cerboni

et al. 2021

Nat Commun

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“…Conventional enrichment strategies that are used by others, such as co-transfection with a fluorescent protein, only report on the efficiency of plasmid delivery to a cell. Along similar lines, as we have previously shown with cytosine base editors (CBEs), reporters of expression in which a fluorescent protein is expressed along with the base editor do not directly report on base-editing activating within a cell [ 16 – 21 ]. To determine if the same was true with adenosine base-editing approaches, we transfected HEK293 cells with a reporter plasmid (mCherry), an adenine base editor (ABEmax; pCMV-ABEmax), and a sgRNA for a genomic target site [sg(TS)] [ 22 ].…”

Section: Resultsmentioning

confidence: 87%

A Cas9-mediated adenosine transient reporter enables enrichment of ABE-targeted cells

et al. 2020

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Background Adenine base editors (ABE) enable single nucleotide modifications without the need for double-stranded DNA breaks (DSBs) induced by conventional CRIPSR/Cas9-based approaches. However, most approaches that employ ABEs require inefficient downstream technologies to identify desired targeted mutations within large populations of manipulated cells. In this study, we developed a fluorescence-based method, named “Cas9-mediated adenosine transient reporter for editing enrichment” (CasMAs-TREE; herein abbreviated XMAS-TREE), to facilitate the real-time identification of base-edited cell populations. Results To establish a fluorescent-based assay able to detect ABE activity within a cell in real time, we designed a construct encoding a mCherry fluorescent protein followed by a stop codon (TGA) preceding the coding sequence for a green fluorescent protein (GFP), allowing translational readthrough and expression of GFP after A-to-G conversion of the codon to “TGG.” At several independent loci, we demonstrate that XMAS-TREE can be used for the highly efficient purification of targeted cells. Moreover, we demonstrate that XMAS-TREE can be employed in the context of multiplexed editing strategies to simultaneous modify several genomic loci. In addition, we employ XMAS-TREE to efficiently edit human pluripotent stem cells (hPSCs), a cell type traditionally resistant to genetic modification. Furthermore, we utilize XMAS-TREE to generate clonal isogenic hPSCs at target sites not editable using well-established reporter of transfection (RoT)-based strategies. Conclusion We established a method to detect adenosine base-editing activity within a cell, which increases the efficiency of editing at multiple genomic locations through an enrichment of edited cells. In the future, XMAS-TREE will greatly accelerate the application of ABEs in biomedical research.

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“…Typically, researchers must screen~500 clones to obtain a single heterozygous mutant, which represents a significant roadblock to many labs [91]. Fortunately, base editors and prime editors introduce SNVs with significantly higher SNV:indel ratios, offering a promising alternative in generating models of population-specific diseases [92]. Furthermore, as our understanding of human genetics expands, we predict that the need to study epistatic traits and haplotypes (i.e., multiple SNVs at a time) will be necessary to fully understand the relationship between genetic variation and traits and disease.…”

Section: Previous Methods For Functional Investigationmentioning

confidence: 99%

Rewriting Human History and Empowering Indigenous Communities with Genome Editing Tools

Fox

Rallapalli

Komor

2020

Genes

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Appropriate empirical-based evidence and detailed theoretical considerations should be used for evolutionary explanations of phenotypic variation observed in the field of human population genetics (especially Indigenous populations). Investigators within the population genetics community frequently overlook the importance of these criteria when associating observed phenotypic variation with evolutionary explanations. A functional investigation of population-specific variation using cutting-edge genome editing tools has the potential to empower the population genetics community by holding “just-so” evolutionary explanations accountable. Here, we detail currently available precision genome editing tools and methods, with a particular emphasis on base editing, that can be applied to functionally investigate population-specific point mutations. We use the recent identification of thrifty mutations in the CREBRF gene as an example of the current dire need for an alliance between the fields of population genetics and genome editing.

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A transient reporter for editing enrichment (TREE) in human cells

Cited by 39 publications

References 59 publications

Universal toxin-based selection for precise genome engineering in human cells

Universal toxin-based selection for precise genome engineering in human cells

A Cas9-mediated adenosine transient reporter enables enrichment of ABE-targeted cells

Rewriting Human History and Empowering Indigenous Communities with Genome Editing Tools

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