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

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

Research output: Contribution to journalArticle

Abstract

BACKGROUND: 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. RESULTS: 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. CONCLUSION: 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.

Original languageEnglish (US)
Number of pages1
JournalBMC genomics
Volume20
Issue number1
DOIs
StatePublished - Jul 24 2019

Fingerprint

Fluorescence
DNA
Proteins
Genome
Clustered Regularly Interspaced Short Palindromic Repeats
Guide RNA
Initiator Codon
Gene Editing
DNA Sequence Analysis
Noise
Nucleotides
Cell Line
Equipment and Supplies
Mutation

Keywords

  • CRIPSR-Cas9
  • Genome editing
  • In-del
  • Insertion-deletion
  • NHEJ
  • Reporter

ASJC Scopus subject areas

  • Biotechnology
  • Genetics

Cite this

Insertion/deletion-activated frame-shift fluorescence protein is a sensitive reporter for genomic DNA editing. / Kumar, Akhilesh; Birnbaum, Michael D.; Moorthy, Balaji T.; Singh, Jayanti; Palovcak, Anna; Patel, Devang M.; Zhang, Fangliang.

In: BMC genomics, Vol. 20, No. 1, 24.07.2019.

Research output: Contribution to journalArticle

Kumar, Akhilesh ; Birnbaum, Michael D. ; Moorthy, Balaji T. ; Singh, Jayanti ; Palovcak, Anna ; Patel, Devang M. ; Zhang, Fangliang. / Insertion/deletion-activated frame-shift fluorescence protein is a sensitive reporter for genomic DNA editing. In: BMC genomics. 2019 ; Vol. 20, No. 1.
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AU - Moorthy, Balaji T.

AU - Singh, Jayanti

AU - Palovcak, Anna

AU - Patel, Devang M.

AU - Zhang, Fangliang

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