Generation and Molecular Characterization of CRISPR/Cas9-Induced Mutations in 63 Immunity-Associated Genes in Tomato Reveals Specificity and a Range of Gene Modifications.

Ning Zhang,Gregory B Martin,Joyce Van Eck,Holly M Roberts

doi:10.3389/fpls.2020.00010

Ning Zhang, Gregory B Martin + Show 2 more

Open Access

https://doi.org/10.3389/fpls.2020.00010

Copy DOI

Abstract

The CRISPR/Cas9 system is a powerful tool for targeted gene editing in many organisms including plants. However, most of the reported uses of CRISPR/Cas9 in plants have focused on modifying one or a few genes, and thus the overall specificity, types of mutations, and heritability of gene alterations remain unclear. Here, we describe the molecular characterization of 361 T0 transgenic tomato plants that were generated using CRISPR/Cas9 to induce mutations in 63 immunity-associated genes. Among the T0 transformed plants, 245 carried mutations (68%), with 20% of those plants being homozygous for the mutation, 30% being heterozygous, 32% having two different mutations (biallelic), and 18% having multiple mutations (chimeric). The mutations were predominantly short insertions or deletions, with 87% of the affected sequences being smaller than 10 bp. The majority of 1 bp insertions were A (50%) or T (29%). The mutations from the T0 generation were stably transmitted to later generations, although new mutations were detected in some T1 plants. No mutations were detected in 18 potential off-target sites among 144 plants. Our study provides a broad and detailed view into the effectiveness of CRISPR/Cas9 for genome editing in an economically important plant species.

Highlights

Derived from a native adaptive immune system in eubacteria and archaea, the CRISPR/Cas system enables the alteration of DNA sequences in many organisms to achieve precise gene modifications (Jaganathan et al, 2018)
To study the efficiency and specificity of genome editing in tomato by CRISPR/Cas9 and to better understand plant-pathogen interactions, we generated a collection of tomato lines with targeted CRISPR/Cas9-induced mutations in genes that have been implicated in the immune response
Our effort to generate a large number of CRISPR/Cas9-induced tomato mutants targeting immunity-associated genes demonstrates that this mutation approach is efficient and robust for gene editing in tomato

Summary

Introduction

Derived from a native adaptive immune system in eubacteria and archaea, the CRISPR/Cas system enables the alteration of DNA sequences in many organisms to achieve precise gene modifications (Jaganathan et al, 2018). (SpCas9) requires the 20-bp spacer sequence of a guide RNA (gRNA) to recognize a complementary target DNA site upstream of a protospacer adjacent motif (PAM) and generates a double-stranded breaks (DSB) near the target region (Xie and Yang, 2013). Compared to other genome editing tools such as zinc finger nucleases (ZFNs; Kim et al, 1996) and transcription activator-like effector nucleases (TALENs; Bogdanove and Voytas, 2011), CRISPR/Cas is more robust in that the Cas protein can theoretically bind to any genomic region preceding a PAM site and, importantly, target multiple sites simultaneously. The most effective way to minimize off-target mutations is to select a gRNA target with little or no homology to other genomic regions

Methods

Results

Conclusion