Genome-wide identification and analysis of highly specific CRISPR/Cas9 editing sites in pepper (Capsicum annuum L.)

Guanliang Li; Ziyan Zhou; Lingrui Liang; Zhao Song; Yafei Hu; Junjie Cui; Weili Chen; Kailin Hu; Jiaowen Cheng

doi:10.1371/journal.pone.0244515

Genome-wide identification and analysis of highly specific CRISPR/Cas9 editing sites in pepper (Capsicum annuum L.)

PLoS One. 2020 Dec 29;15(12):e0244515. doi: 10.1371/journal.pone.0244515. eCollection 2020.

Authors

Guanliang Li^{1

2

3}, Ziyan Zhou^{1

2

3}, Lingrui Liang^{1

2

3}, Zhao Song^{1

2

3}, Yafei Hu^{1

2

3}, Junjie Cui⁴, Weili Chen^{1

2

3}, Kailin Hu^{1

2

3}, Jiaowen Cheng^{1

2

3}

Affiliations

¹ College of Horticulture, South China Agricultural University, Guangzhou, China.
² South China Key Laboratory of Urban Agriculture, Ministry of Agriculture and Rural Affairs, Guangzhou, China.
³ Key Laboratory of Biology and Genetic Improvement of Horticultural Crops (South China), Ministry of Agriculture and Rural Affairs, Guangzhou, China.
⁴ Department of Horticulture, College of Food Science and Engineering, Foshan University, Foshan, Guangdong, China.

Abstract

The CRISPR/Cas9 system is an efficient genome editing tool that possesses the outstanding advantages of simplicity and high efficiency. Genome-wide identification and specificity analysis of editing sites is an effective approach for mitigating the risk of off-target effects of CRISPR/Cas9 and has been applied in several plant species but has not yet been reported in pepper. In present study, we first identified genome-wide CRISPR/Cas9 editing sites based on the 'Zunla-1' reference genome and then evaluated the specificity of CRISPR/Cas9 editing sites through whole-genome alignment. Results showed that a total of 603,202,314 CRISPR/Cas9 editing sites, including 229,909,837 (~38.11%) NGG-PAM sites and 373,292,477 (~61.89%) NAG-PAM sites, were detectable in the pepper genome, and the systematic characterization of their composition and distribution was performed. Furthermore, 29,623,855 highly specific NGG-PAM sites were identified through whole-genome alignment analysis. There were 26,699,38 (~90.13%) highly specific NGG-PAM sites located in intergenic regions, which was 9.13 times of the number in genic regions, but the average density in genic regions was higher than that in intergenic regions. More importantly, 34,251 (~96.93%) out of 35,336 annotated genes exhibited at least one highly specific NGG-PAM site in their exons, and 90.50% of the annotated genes exhibited at least 4 highly specific NGG- PAM sites, indicating that the set of highly specific CRISPR/Cas9 editing sites identified in this study was widely applicable and conducive to the minimization of the off-target effects of CRISPR/Cas9 in pepper.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

CRISPR-Cas Systems / genetics*
Capsicum / genetics*
Gene Editing / methods*
Genome, Plant / genetics
Plant Breeding
Sequence Alignment

Associated data

figshare/10.6084/m9.figshare.13324211
figshare/10.6084/m9.figshare.13345238

Grants and funding

This work was supported by the Guangzhou Science and Technology Plan Project (201704020019), the National Natural Science Foundation of China (31701921, 31672162) and the Guangdong Basic and Applied Basic Research Foundation (2019A1515011784). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.