Application of CRISPR/Cas9 technology in wild apple (Malus sieverii) for paired sites gene editing

Yan Zhang,Tohir A Bozorov,Ping Zhou,Daoyuan Zhang

doi:10.1186/s13007-021-00769-8

Abstract

BackgroundXinjiang wild apple is an important tree of the Tianshan Mountains, and in recent years, it has undergone destruction by many biotic and abiotic stress and human activities. It is necessary to use new technologies to research its genomic function and molecular improvement. The clustered regulatory interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) system has been successfully applied to genetic improvement in many crops, but its editing capability varies depending on the different combinations of the synthetic guide RNA (sgRNA) and Cas9 protein expression devices.ResultsIn this study, we used 2 systems of vectors with paired sgRNAs targeting to MsPDS. As expected, we successfully induced the albino phenotype of calli and buds in both systems.ConclusionsWe conclude that CRISPR/Cas9 is a powerful system for editing the wild apple genome and expands the range of plants available for gene editing.

Highlights

Xinjiang wild apple is an important tree of the Tianshan Mountains, and in recent years, it has undergone destruction by many biotic and abiotic stress and human activities
Constructs for the targeted genome editing of wild apple To edit the target gene, we selected two different systems, pYL-clustered regulatory interspaced short palindromic repeats (CRISPR)/Cas9 and pTG-CRISPR/Cas9, and constructed 8 different plasmids harbouring 2 synthetic guide RNA (sgRNA) according to their methods (Fig. 1C) [33, 40, 41]
For the pYL system, Cas9 endonuclease was under the maize ubiquitin promoter (PUBI), and a sgRNA expression cassette was under the AtU3d promoter

Summary

Introduction

Xinjiang wild apple is an important tree of the Tianshan Mountains, and in recent years, it has undergone destruction by many biotic and abiotic stress and human activities. Many genetic studies have shown that the wild apple M. sieversii is the Genome editing tools include zinc-finger nucleases (zinc-fingernuclease), transcription activator-like effector nuclease (TALEN), and the clustered regulatory interspaced short palindromic repeats (CRISPR)/CRISPRassociated protein (Cas) system (CRISPR/Cas) [10]. Type II Cas protein has been widely adopted as a simple and highly efficient targeted genome editing tool for multiplex genome engineering using CRISPR/ Cas systems [12]. In this system, the mature dual CRISPR RNA consists of a crRNA and small trans-activating CRISPR RNA (tracrRNA), which forms a functional complex with the endonuclease Cas. This is the first successful application of the CRISPR/Cas gene editing system in this species, which will provide strong technical support for its gene function research, utilization, and conservation

Methods

Results

Discussion

Conclusion