Plant Molecular Biology

, Volume 82, Issue 3, pp 207–221 | Cite as

A rapid assay to quantify the cleavage efficiency of custom-designed nucleases in planta

  • Ross A. Johnson
  • Vyacheslav Gurevich
  • Avraham A. Levy
Article

Abstract

Custom-designed nucleases are a promising technology for genome editing through the catalysis of double-strand DNA breaks within target loci and subsequent repair by the host cell, which can result in targeted mutagenesis or gene replacement. Implementing this new technology requires a rapid means to determine the cleavage efficiency of these custom-designed proteins in planta. Here we present such an assay that is based on cleavage-dependent luciferase gene correction as part of a transient dual-luciferase® reporter (Promega) expression system. This assay consists of co-infiltrating Nicotiana benthamiana leaves with two Agrobacterium tumefaciens strains: one contains the target sequence embedded within a luciferase reporter gene and the second strain contains the custom-designed nuclease gene(s). We compared repair following site-specific nuclease digestion through non-homologous DNA end-joining, as opposed to single strand DNA annealing, as a means to restore an out-of-frame luciferase gene cleavage-reporter construct. We show, using luminometer measurements and bioluminescence imaging, that the assay for non-homologous end-joining is sensitive, quantitative, reproducible and rapid in estimating custom-designed nucleases’ cleavage efficiency. We detected cleavage by two out of three transcription activator-like effector nucleases that we custom-designed for targets in the Arabidopsis CRUCIFERIN3 gene, and we compared with the well-established ‘QQR’ zinc-finger nuclease. The assay we report requires only standard equipment and basic plant molecular biology techniques, and it can be carried out within a few days. Different types of custom-designed nucleases can be preliminarily tested in our assay system before their downstream application in plant genome editing.

Keywords

DNA repair Genome engineering Non-homologous end-joining TALENs Targeted mutagenesis ZFNs 

Abbreviations

ΔG

Gibbs energy values for DNA secondary structures

35S

Cauliflower mosaic virus-derived 35S promoter

AtCRU3

Arabidopsis thaliana CRUCIFERIN3 gene

Cps

Counts per second

F/shift, fs

A translational reading frame-shift mutation

Kb

Kilo base pair(s)

LUC

Gene encoding a luciferase enzyme derived from Photinus pyralis

NHEJ

Non-homologous end-joining

QQR

The name given to a previously-reported zinc-finger nuclease

ORF

Translational open reading frame

Recog. site

The DNA site recognized by a custom-designed nuclease

REN

The gene encoding a luciferase enzyme derived from Renilla reniformis

SSA

Single-strand annealing

Sc

A premature stop codon

T

A transcription ‘terminator’ sequence

T-DNA

A. tumefaciens transfer-DNA

TALEN

Transcription activator-like effector nuclease

YFP

Yellow fluorescent protein

ZFN

Zinc-finger nuclease

Supplementary material

11103_2013_52_MOESM1_ESM.pdf (36 kb)
Online Resource 1 The sequences and applications of the oligonucleotide primer sequences that were used in the study. Supplementary material 1 (PDF 36 kb)
11103_2013_52_MOESM2_ESM.pdf (136 kb)
Online Resource 2 Assays for site-specific cleavage-induced NHEJ in response to QQR ZFN expression, which complements Fig. 2. Supplementary material 2 (PDF 136 kb)
11103_2013_52_MOESM3_ESM.pdf (21 kb)
Online Resource 3 The luciferase data from assays for NHEJ with the QQR ZFN, which complements Fig. 2 and Online Resource 2.Supplementary material 3 (PDF 22 kb)
11103_2013_52_MOESM4_ESM.pdf (93 kb)
Online Resource 4 The luciferase data from testing reporter signal levels arising from SSA, versus NHEJ, for the QQR ZFN, which complements Fig. 3.Supplementary material 4 (PDF 94 kb)
11103_2013_52_MOESM5_ESM.pdf (28 kb)
Online Resource 5 The luciferase data from comparing the cleavage efficiency of TALENs recognizing the AtCRU3 gene at T494, T852 and T1461 sites, which complements Fig. 6.Supplementary material 5 (PDF 28 kb)
11103_2013_52_MOESM6_ESM.pdf (34 kb)
Online Resource 6 The luciferase data from comparing two TALEN homodimers and their heterodimer that was custom-designed for T494 within the AtCRU3 gene, which complements Fig. 7.Supplementary material 6 (PDF 34 kb)
11103_2013_52_MOESM7_ESM.pdf (141 kb)
Online Resource 7 Quantification of firefly-derived luciferase activity in imaged leaves, which complements Fig. 8.Supplementary material 7 (PDF 141 kb)

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Copyright information

© Springer Science+Business Media Dordrecht 2013

Authors and Affiliations

  • Ross A. Johnson
    • 1
  • Vyacheslav Gurevich
    • 1
  • Avraham A. Levy
    • 1
  1. 1.Department of Plant Sciencesthe Weizmann Institute of ScienceRehovotIsrael

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