Efficient CRISPR/Cas9 genome editing with Citrus embryogenic cell cultures

Manjul Dutt,Sameena E Tanwir,Zhonglin Mou,Xudong Zhang,Jude W Grosser

doi:10.1186/s12896-020-00652-9

Manjul Dutt, Sameena E Tanwir + Show 3 more

Open Access

https://doi.org/10.1186/s12896-020-00652-9

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Abstract

BackgroundDevelopment of precise genome editing strategies is a prerequisite for producing edited plants that can aid in the study of gene function and help understand the genetic traits in a cultivar. Citrus embryogenic cell cultures can be used to rapidly produce a large population of genome edited transformed citrus lines. The ability to introduce specific mutations in the genome of these cells using two constructs (pC-PDS1 and pC-PDS2) was evaluated in this study.ResultsCitrus sinensis ‘EV2’ embryogenic cell cultures are amenable to Agrobacterium-mediated CRISPR/Cas9-based genome editing. Guide RNAs (gRNAs) targeting two locations in the phytoene desaturase (PDS) gene were either driven by the Arabidopsis U6–26 promoter (pC-PDS1) or assembled as a Csy4 array under the control of the CmYLCV promoter (pC-PDS2). All transgenic embryos were completely albino and no variegated phenotype was observed. We evaluated 12 lines from each construct in this study and the majority contain either insertion (1–2 bp), substitution (1 bp), or deletion (1–3 bp) mutations that occurred close to the protospacer adjacent motif.ConclusionsBoth the pC-PDS1 and pC-PDS2 could successfully edit the citrus embryogenic cell cultures. However, the editing efficiency was dependent on the gRNA, confirming that the selection of a proper gRNA is essential for successful genome editing using the CRISPR/Cas9 technique. Also, utilization of embryogenic cell cultures offers another option for successful genome editing in citrus.

Highlights

Development of precise genome editing strategies is a prerequisite for producing edited plants that can aid in the study of gene function and help understand the genetic traits in a cultivar
To fully expand the potential of the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system in citrus, we evaluated a CRISPR construct that encoded guide Ribonucleic Acid (RNA) (gRNA) driven by the Arabidopsis U6–26 pol RNA polymerase III (III) promoter and compared it to a construct that harnessed the Csy4 bacterial endoribonuclease's RNA processing ability
The two distinct gRNAs used in this study were based on earlier published reports

Summary

Introduction

Development of precise genome editing strategies is a prerequisite for producing edited plants that can aid in the study of gene function and help understand the genetic traits in a cultivar. Considerable efforts have been made to develop superior citrus cultivars that can better withstand abiotic and biotic stresses and at the same time produce optimum yields using conventional breeding and genetic modification strategies. CRISPR/Cas has emerged as the most extensively used genome-editing system in the recent years [6, 7]. In addition to the pioneering studies in Arabidopsis [8] and Nicotiana [9,10,11], the CRISPR/Cas system has been successfully used to target specific genomic sequences of interest for the development of genome edited citrus [12,13,14,15,16]

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