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Plant Biotechnol J. 2019 Feb;17(2):362-372. doi: 10.1111/pbi.12982. Epub 2018 Jul 22.

Activities and specificities of CRISPR/Cas9 and Cas12a nucleases for targeted mutagenesis in maize.

Author information

1
Crop Bioengineering Center, Iowa State University, Ames, IA, USA.
2
Department of Agronomy, Iowa State University, Ames, IA, USA.
3
Department of Plant Science and Landscape Architecture, University of Maryland, College Park, MD, USA.
4
Molecular Pathology Unit, Center for Cancer Research, and Center for Computational and Integrative Biology, Massachusetts General Hospital, Charlestown, MA, USA.
5
Department of Pathology, Harvard Medical School, Boston, MA, USA.
6
Department of Biostatistics, Harvard T.H. Chan School of Public Health, Boston, MA, USA.
7
Department of Genetics, Development and Cell Biology, Iowa State University, Ames, IA, USA.
8
Bioinformatics and Computational Biology Program, Iowa State University, Ames, IA, USA.
9
Institute for Bioscience and Biotechnology Research, University of Maryland, Rockville, MD, USA.

Abstract

CRISPR/Cas9 and Cas12a (Cpf1) nucleases are two of the most powerful genome editing tools in plants. In this work, we compared their activities by targeting maize glossy2 gene coding region that has overlapping sequences recognized by both nucleases. We introduced constructs carrying SpCas9-guide RNA (gRNA) and LbCas12a-CRISPR RNA (crRNA) into maize inbred B104 embryos using Agrobacterium-mediated transformation. On-target mutation analysis showed that 90%-100% of the Cas9-edited T0 plants carried indel mutations and 63%-77% of them were homozygous or biallelic mutants. In contrast, 0%-60% of Cas12a-edited T0 plants had on-target mutations. We then conducted CIRCLE-seq analysis to identify genome-wide potential off-target sites for Cas9. A total of 18 and 67 potential off-targets were identified for the two gRNAs, respectively, with an average of five mismatches compared to the target sites. Sequencing analysis of a selected subset of the off-target sites revealed no detectable level of mutations in the T1 plants, which constitutively express Cas9 nuclease and gRNAs. In conclusion, our results suggest that the CRISPR/Cas9 system used in this study is highly efficient and specific for genome editing in maize, while CRISPR/Cas12a needs further optimization for improved editing efficiency.

KEYWORDS:

Zea mays ; CIRCLE-seq; CRISPR/Cas; Cas12a (Cpf1); genome editing; off-target

PMID:
29972722
PMCID:
PMC6320322
DOI:
10.1111/pbi.12982
[Indexed for MEDLINE]
Free PMC Article

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