Identification of Transgene-Free CRISPR-Edited Plants of Rice, Tomato, and Arabidopsis by Monitoring DsRED Fluorescence in Dry Seeds.

Norma Aliaga-Franco,Anjil K Srivastava,Cunjin Zhang,Antonio Granell,Eugenio G Minguet,Ari Sadanandom,David Alabadí,Silvia Presa,Miguel A Blázquez

doi:10.3389/fpls.2019.01150

Abstract

Efficient elimination of the editing machinery remains a challenge in plant biotechnology after genome editing to minimize the probability of off-target mutations, but it is also important to deliver end users with edited plants free of foreign DNA. Using the modular cloning system Golden Braid, we have included a fluorescence-dependent transgene monitoring module to the genome-editing tool box. We have tested this approach in Solanum lycopersicum, Oryza sativa, and Arabidopsis thaliana. We demonstrate that DsRED fluorescence visualization works efficiently in dry seeds as marker for the detection of the transgene in the three species allowing an efficient method for selecting transgene-free dry seeds. In the first generation of DsRED-free CRISPR/Cas9 null segregants, we detected gene editing of selected targets including homozygous mutants for the plant species tested. We demonstrate that this strategy allows rapid selection of transgene-free homozygous edited crop plants in a single generation after in vitro transformation.

Highlights

CRISPR/Cas technology, adapted from bacterial immune system (Bolotin et al, 2005; Mojica et al, 2005), for specific and precise modification of genomes (Jinek et al, 2012; Wiedenheft et al, 2012) has transformed molecular biology
As a proof of concept, we decided to use the gene encoding IAA methyl transferase (IAMT) as a gene-editing target in three plant model species (A. thaliana, O. sativa, and S. lycopersicum), given that loss of function results only in difficulty for hypocotyl reorientation after gravistimulation (Abbas et al, 2018b) and increased pollen tube growth rate (Abbas et al, 2018a), neither of which are traits that can bias our identification of mutations by direct observation unless specific tests are performed
Those primary transformant plants contain one copy of the transgene that will be segregated in the generation independently of any CRISPR-induced mutations in germline; we could use DsRED visualization as marker of transgene presence to select nonfluorescent seeds and search for mutations

Summary

Introduction

CRISPR/Cas technology, adapted from bacterial immune system (Bolotin et al, 2005; Mojica et al, 2005), for specific and precise modification of genomes (Jinek et al, 2012; Wiedenheft et al, 2012) has transformed molecular biology. Specific resistance genes were introduced into each vector for in vitro selection, as required by crop transformation protocols: hygromycin (GB0211) and kanamycin (GB0226) for rice and tomato, respectively.

Results

Conclusion