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/gkaa027

Cited by 10 publications

(19 citation statements)

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“…One solution for increasing the e ciency of base editing without generating nick-induced indels would be the enrichment of those cells that contain dABE-and dCBE-edited bases, using a marker. Unfortunately, the markers that have been developed so far to enrich CBE-or ABE-edited cells exclusively employ nickase SpCas9 12,13,14,15,16,17 . One of the objectives of our study has been to develop a marker that is sensitive enough to enable high e ciency base editing by enriching dABE-and dCBE-edited cells, without intentionally nicking the DNA.…”

Section: Introductionmentioning

confidence: 99%

“…These assays are based on the installation or alteration of a start or stop codon 16,17 , or they rescue a disruptive amino acid and concomitantly recover a uorescent signal 14 . Alternatively, a non-synonymous mutation in the chromophore of a uorescent protein that induces uorescence spectral change has also been explored as an option to monitor base editing activity 12,15 . Although these assays exploit clever strategies, they are limited to only a few target sequences or exhibit high background signal and/or have relatively low sensitivity 12,13,14,15,16 .…”

Section: Introductionmentioning

confidence: 99%

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BEAR reveals that increased fidelity variants can successfully reduce the mismatch-tolerance of adenine but not cytosine base editors

Tálas

Simon

Kulcsár

et al. 2020

Preprint

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Adenine and cytosine base editors (ABE, CBE) are designed to generate single base mutations in genes without necessarily generating DNA double-strand breaks and undesired indel mutations. However, the activity of base editors employing an inactive (dead) SpCas9 is generally low, which may be increased only at the expense of generating undesired indels by using a nickase SpCas9. We have increased the efficiency of dead base editors to a level comparable to that of nickase base editors by enriching cells labelled for efficient base editing using Base Editor Activity Reporter (BEAR), a plasmid-based, fluorescent tool. Furthermore, by exploiting the semi-high-throughput potential of BEAR, we have examined the applicability of increased fidelity variants to decrease Cas9-dependent off-target effects that revealed that CBE remains active on off-targets where increased fidelity mutations and/or mismatches decrease the activity of ABE, making the strategy of applying increased fidelity variants more beneficial for ABE than for CBE.

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Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

BEAR reveals that increased fidelity variants can successfully reduce the mismatch-tolerance of adenine but not cytosine base editors

Tálas

Simon

Kulcsár

et al. 2020

Preprint

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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 baseediting activating within a cell [16][17][18][19][20][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: Development Of a Fluorescent Reporter For Cas9-mediated Adenmentioning

confidence: 66%

“…That said, BEs can be used in the context of cellular models of human disease models to establish genotypeto-phenotype relationship associated with genetic risk factors, investigate disease mechanisms, and test therapeutic strategies. In our previous work, we describe the development of a transient reporter for editing enrichment (TREE) as a fluorescence-based assay to report on cytosine base-editing (CBE) activity within a single cell [16]. In this work, we develop an analogous reporter system, Cas9-mediated adenosine transient reporter for editing enrichment (CasMAs-TREE; XMAS-TREE) that allows for the real-time detection of adenosine base editing (ABE) for the identification and enrichment of base-edited cell populations.…”

Section: Discussionmentioning

confidence: 99%

“…Along similar lines, previous work has shown that other factors including intracellular formation of Cas9/sgRNA complexes, cell cycle, chromatin accessibility, local epigenetic modifications, and sequence context influence editing efficiency [23]. To that end, we sought to leverage our experience developing fluorescent reporters of editing activity to enable XMAS-TREE [16,21]. To establish a fluorescent assay to detect ABE activity within a cell, we engineered a construct encoding a mCherry fluorescent protein followed by a stop codon (TGA) immediately preceding the coding sequence for a green fluorescent protein (GFP).…”

Section: Development Of a Fluorescent Reporter For Cas9-mediated Adenmentioning

confidence: 97%

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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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Base editing-coupled survival screening enabled high-sensitive analysis of PAM compatibility and finding of the new possible off-target

Guo

Zheng

et al. 2021

iScience

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Base editing (BE) is a promising genome engineering tool for modifying DNA or RNA and has been widely used in various microorganisms as well as eukaryotic cells. Despite the proximal protospacer adjacent motif (PAM) is critical to the targeting range and off-target effect of BE, there is still lack of a specific approach to analyze the PAM pattern in BE systems. Here, we developed a base editingcoupled survival screening method. Using dCas9 from Streptococcus pyogenes (SpdCas9) and its variants xdCas9 3.7 and dCas9 NG as example, their PAM patterns in BE systems were extensively characterized using the NNNN PAM library with high sensitivity. In addition to the typical PAM recognition features, we observed more unique PAMs exhibiting BE activity. These PAM patterns will boost the finding of potential off-target editing event arising from non-canonical PAMs and provide the guidelines for PAM usage in the BE system.

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

Cited by 10 publications

References 0 publications

BEAR reveals that increased fidelity variants can successfully reduce the mismatch-tolerance of adenine but not cytosine base editors

BEAR reveals that increased fidelity variants can successfully reduce the mismatch-tolerance of adenine but not cytosine base editors

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

Base editing-coupled survival screening enabled high-sensitive analysis of PAM compatibility and finding of the new possible off-target

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