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

Autor: Lee K; Crop Bioengineering Center, Iowa State University, Ames, IA, USA.; Department of Agronomy, Iowa State University, Ames, IA, USA., Zhang Y; Department of Plant Science and Landscape Architecture, University of Maryland, College Park, MD, USA., Kleinstiver BP; Molecular Pathology Unit, Center for Cancer Research, and Center for Computational and Integrative Biology, Massachusetts General Hospital, Charlestown, MA, USA.; Department of Pathology, Harvard Medical School, Boston, MA, USA., Guo JA; Molecular Pathology Unit, Center for Cancer Research, and Center for Computational and Integrative Biology, Massachusetts General Hospital, Charlestown, MA, USA., Aryee MJ; Department of Pathology, Harvard Medical School, Boston, MA, USA.; Department of Biostatistics, Harvard T.H. Chan School of Public Health, Boston, MA, USA., Miller J; Crop Bioengineering Center, Iowa State University, Ames, IA, USA., Malzahn A; Department of Plant Science and Landscape Architecture, University of Maryland, College Park, MD, USA., Zarecor S; Crop Bioengineering Center, Iowa State University, Ames, IA, USA.; Department of Genetics, Development and Cell Biology, Iowa State University, Ames, IA, USA., Lawrence-Dill CJ; Crop Bioengineering Center, Iowa State University, Ames, IA, USA.; Department of Agronomy, Iowa State University, Ames, IA, USA.; Department of Genetics, Development and Cell Biology, Iowa State University, Ames, IA, USA.; Bioinformatics and Computational Biology Program, Iowa State University, Ames, IA, USA., Joung JK; Molecular Pathology Unit, Center for Cancer Research, and Center for Computational and Integrative Biology, Massachusetts General Hospital, Charlestown, MA, USA.; Department of Pathology, Harvard Medical School, Boston, MA, USA., Qi Y; Department of Plant Science and Landscape Architecture, University of Maryland, College Park, MD, USA.; Institute for Bioscience and Biotechnology Research, University of Maryland, Rockville, MD, USA., Wang K; Crop Bioengineering Center, Iowa State University, Ames, IA, USA.; Department of Agronomy, Iowa State University, Ames, IA, USA.
Jazyk: angličtina
Zdroj: Plant biotechnology journal [Plant Biotechnol J] 2019 Feb; Vol. 17 (2), pp. 362-372. Date of Electronic Publication: 2018 Jul 22.
DOI: 10.1111/pbi.12982
Abstrakt: 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.
(© 2018 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.)
Databáze: MEDLINE
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