Rapid and user-friendly open-source CRISPR/Cas9 system for single- or multi-site editing of tomato genome

Nan Hu,Zhiqiang Xian,Yudong Liu,Ning Li,Deding Su,Wei Huang,Zhengguo Li,Fang Yan,Jingxuan Chen

doi:10.1038/s41438-018-0082-6

Abstract

CRISPR/Cas9-induced genome editing is a powerful tool for studying gene function in a variety of organisms, including plants. Using multi-sgRNAs to target one or more genes is helpful to improve the efficacy of gene editing and facilitate multi-gene editing. Here, we describe a CRISPR/Cas9 system which can be conveniently developed as a CRISPR kit. SgRNA expression cassettes can be rapidly generated by one-step PCR using our CRISPR kit. In our kit, there are two binary vectors pHNCas9 and pHNCas9HT. The binary vector pHNCas9 was constructed to allow to assemble up to eight sgRNA expression cassettes by one-step Golden Gate cloning. Another binary vector pHNCas9HT can be used to generate a large number of single target constructs by directly transforming ligation reactions products into A. tumefaciens without several procedures, such as PCR and plasmid extraction. The two binary vectors are designed according to the principles of standard BioBrick assembly to be used as an open-source tool. For example, we used BioBrick as a visual T-DNA tag. We also developed a primer design aid to complement the system. With this primer design aid, researchers can rapidly obtain primers and GC content, and sgRNA sequence of target site. Our CRISPR/Cas9 system can perform single- and multi-site editing and multiple gene editing to produce various types of mutations in tomato. This rapid and user-friendly CRISPR/Cas9 system for tomato can be potentially used for mutagenesis of important crop species for genetic improvement and is suitable for research into the function of genes.

Highlights

Reverse genetics is a fundamental way of studying gene function in plants, where a gene of interest is knocked out or its expression is attenuated to deduce the normal function of the gene from the observed phenotypic effects[1]
We have developed a Cas[9] system containing two binary vectors pHNCas[9] and pHNCas9HT. pHNCas9HT can be used for the construction of sgRNA expression cassettes without PCR and direct transformation of Agrobacterium tumefaciens
We have developed an efficient, user-friendly, potentially high-throughput, open-source CRISPR/ Cas[9] system optimized for targeting multiple genes and genomic sites in tomato

Summary

Introduction

Reverse genetics is a fundamental way of studying gene function in plants, where a gene of interest is knocked out or its expression is attenuated to deduce the normal function of the gene from the observed phenotypic effects[1]. Classical reverse genetic strategies to target genes of interest include antisense inhibition, virus-induced gene silencing, and post-transcriptional gene silencing (PTGS) based on double-stranded RNA, either driven by small interfering RNA or artificial micro RNAs1–4. A drawback of PTGS is its instability from tissue to tissue or generation to generation, as the small RNAs can are known. This approach is not applicable in most plant species[6]. Two protein-based DNA targeting systems, zinc-finger nucleases (ZFNs), and

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