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Insertion/deletion-activated frame-shift fluorescence protein is a sensitive reporter for genomic DNA editing

Overview of attention for article published in BMC Genomics, July 2019
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Title
Insertion/deletion-activated frame-shift fluorescence protein is a sensitive reporter for genomic DNA editing
Published in
BMC Genomics, July 2019
DOI 10.1186/s12864-019-5963-z
Pubmed ID
Authors

Akhilesh Kumar, Michael D. Birnbaum, Balaji T. Moorthy, Jayanti Singh, Anna Palovcak, Devang M. Patel, Fangliang Zhang

Abstract

Reporter methods to quantitatively measure the efficiency and specificity of genome editing tools are important for the development of novel editing techniques and successful applications of available ones. However, the existing methods have major limitations in sensitivity, accuracy, and/or readiness for in vivo applications. Here, we aim to develop a straight-forward method by using nucleotide insertion/deletion resulted from genome editing. In this system, a target sequence with frame-shifting length is inserted after the start codon of a cerulean fluorescence protein (CFP) to inactivate its fluorescence. As such, only a new insertion/deletion event in the target sequence will reactivate the fluorescence. This reporter is therefore termed as "Insertion/deletion-activated frame-shift fluorescence protein". To increase its traceability, an internal ribosome entry site and a red fluorescence protein mCherryFP are placed downstream of the reporter. The percentage of CFP-positive cells can be quantified by fluorescence measuring devices such as flow cytometer as the readout for genome editing frequency. To test the background noise level, sensitivity, and quantitative capacity of this new reporter, we applied this approach to examine the efficiency of genome editing of CRISPR/Cas9 on two different targeting sequences and in three different cell lines, in the presence or absence of guide-RNAs with or without efficiency-compromising mutations. We found that the insertion/deletion-activated frame-shift fluorescence protein has very low background signal, can detect low-efficiency genome editing events driven by mutated guideRNAs, and can quantitatively distinguish genome editing by normal or mutated guideRNA. To further test whether the positive editing event detected by this reporter indeed correspond to genuine insertion/deletion on the genome, we enriched the CFP-positive cells to examine their fluorescence under confocal microscope and to analyze the DNA sequence of the reporter in the genome by Sanger sequencing. We found that the positive events captured by this reporter indeed correlates with genuine DNA insertion/deletion in the expected genome location. The insertion/deletion-activated frame-shift fluorescence protein reporter has very low background, high sensitivity, and is quantitative in nature. It will be able to facilitate the development of new genome editing tools as well as the application of existing tools.

Twitter Demographics

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Mendeley readers

The data shown below were compiled from readership statistics for 3 Mendeley readers of this research output. Click here to see the associated Mendeley record.

Geographical breakdown

Country Count As %
Unknown 3 100%

Demographic breakdown

Readers by professional status Count As %
Researcher 2 67%
Unknown 1 33%
Readers by discipline Count As %
Chemical Engineering 1 33%
Agricultural and Biological Sciences 1 33%
Unknown 1 33%

Attention Score in Context

This research output has an Altmetric Attention Score of 1. This is our high-level measure of the quality and quantity of online attention that it has received. This Attention Score, as well as the ranking and number of research outputs shown below, was calculated when the research output was last mentioned on 26 July 2019.
All research outputs
#12,130,642
of 13,668,760 outputs
Outputs from BMC Genomics
#6,965
of 7,961 outputs
Outputs of similar age
#205,811
of 247,025 outputs
Outputs of similar age from BMC Genomics
#7
of 9 outputs
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