Efficiency and Inheritance of Targeted Mutagenesis in Maize Using CRISPR-Cas9

State Key Laboratory of Agrobiotechnology and National Maize Improvement Center, Department of Plant Genetics and Breeding, China Agricultural University, Beijing 100193, China

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Corresponding author: E-mail address: jlai@cau.edu.cn (Jinsheng Lai)

These authors contributed equally to this work.

摘要

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Abstract: CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated proteins) is an adaptive immune system in bacteria and archaea to defend against invasion from foreign DNA fragments. Recently, it has been developed as a powerful targeted genome editing tool for a wide variety of species. However, its application in maize has only been tested with transiently expressed somatic cells or with a limited number of stable transgenic T₀ plants. The exact efficiency and specificity of the CRISPR/Cas system in the highly complex maize genome has not been documented yet. Here we report an extensive study of the well-studied type II CRISPR-Cas9 system for targeted genome editing in maize, with the codon-optimized Cas9 protein and the short non-coding guide RNA generated through a functional maize U6 snRNA promoter. Targeted gene mutagenesis was detected for 90 loci by maize protoplast assay, with an average cleavage efficiency of 10.67%. Stable knockout transformants for maize phytoene synthase gene (PSY1) were obtained. Mutations occurred in germ cells can be stably inherited to the next generation. Moreover, no off-target effect was detected at the computationally predicted putative off-target loci. No significant difference between the transcriptomes of the Cas9 expressed and non-expressed lines was detected. Our results confirmed that the CRISPR-Cas9 could be successfully applied as a robust targeted genome editing system in maize.
- CRISPR-Cas9 /
- Targeted mutagenesis /
- Heritability /
- Maize
These authors contributed equally to this work.
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